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The only light source for chickens in environmentally controlled houses is an artificial one. Thus, source, spectra, intensity and regimen of light supplementation became major factors in modern meat type bird management. Light spectra affect growth in meat type birds both in ovo and post hatch. Broilers photostimulated in ovo with green light gained significantly more weight than birds incubated under dark conditions. Furthermore, we defined the cellular and molecular events associated with the effect of in ovo green photostimulation on muscle growth. We found that in ovo photostimulation have a stimulatory effect on the proliferation and differentiation of satellite cells and a promoting effect on the uniformity of the muscle fibers in the early post-hatch period. How does in ovo photostimulation affect intracellular events, such as proliferation and differentiation of muscle cells, leading to post-hatch muscle growth? It is possible that the monochromatic green light penetrates the eggshell and has a direct effect on the embryo's muscle. We were unable to detect any proliferative effect of monochromatic green light on cultured myoblasts derived from standard (un-illuminated) E17 embryos and 3-day-old chicks. A more likely explanation is that green light indirectly affects myoblast proliferation by activating the endocrine system; the latter receives photic cues from the retinal or extra-retinal photoreceptors. We gathered some evidence to support these findings; we have shown a higher expression of growth hormone (GH) receptor mRNA in satellite cells derived from green light illuminated chicks. In addition, plasma GH levels and IGF-I levels in muscle tissue, were higher in the green group relative to the dark one in early post-hatch. Another possible explanation for this phenomenon could be that growth factor secretion is activated in response to green light photostimulation. Both retinal and extra-retinal photoreceptors are active during embryogenesis and can be first detected at E14. Combinations of in ovo and post-hatch green light photostimulation to broilers and turkeys did not cause synergetic effect on growth. In a recent study, we found that in ovo green light photostimulation suppresses the green and red opsin receptors gene expression in the last three days before hatching, while red light enhances their expression. Furthermore, we found that the down-regulation of the green and red opsins in response to incubation under monochromatic green lighting lasted up to 9days post hatch, suggesting a possible epigenetic effect.

Melanopsin (OPN4) is a photoreceptive molecule regulating circadian systems in mammals. Previous studies from our laboratory have shown that co-localized dopamine-melatonin (DA-MEL) neurons in the hypothalamic premammillary nucleus (PMM) are putatively photosensitive and exhibit circadian rhythms in DAergic and MELergic activities. This study investigates turkey OPN4x (tOPN4x) mRNA distribution in the hypothalamus and brainstem, and characterizes its expression in PMM DA-MEL neurons, using in situ hybridization (ISH), immunocytochemistry (ICC), double-label ISH/ICC, and real time-PCR. The mRNA encoding tOPN4x was found in anatomically discrete areas in or near the hypothalamus and the brainstem, including nucleus preopticus medialis (POM), nucleus septalis lateralis (SL), PMM and the pineal gland. Double ICC, using tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis)-and OPN4x antibodies, confirmed the existence of OPN4x protein in DA-MEL neurons. Also, tOPN4x mRNA expression was verified with double ISH/ICC using tOPN4x mRNA and TH immunoreactivity. PMM and pineal gland tOPN4x mRNA expression levels were diurnally high during the night and low during the day. A light pulse provided to short day photosensitive hens during the photosensitive phase at night significantly down-regulated tOPN4x expression. The expression level of tOPN4x mRNA in PMM DA-MEL neurons of photorefractory hens was significantly lower as compared with that of short or long day photosensitive hens. The results implicate tOPN4x in hypothalamic PMM DA-MEL neurons as an important component of the photoreceptive system regulating reproductive activity in temperate zone birds.

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual-oscillation in dopamine (DA)/melatonin (MEL) as well as c-fos mRNA expression that is associated with the daily photo-inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase-related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase-delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light-induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase-shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.

Serotonin and catecholamines (dopamine, norepinephrine, epinephrine) have important roles as neurotransmitters in avian reproduction, but their anatomical relationship to the neuroendocrine circuitry that regulates reproduction is poorly understood. Our previous studies have shown that co-localised dopamine-melatonin (DA-MEL) neurones in the avian premammillary nucleus (PMM) are active during periods of photoresponsiveness and, therefore, are potentially photosensitive neurones. Because serotonergic and catecholaminergic neurotransmitters are important regulators of reproductive function in the female turkey, we hypothesised that the serotonergic/catecholaminergic neurones within the brainstem might interact with PMM DA-MEL neurones and constitute an important circuit for reproductive function. To examine this possible interaction, the retrograde fluorescent tract tracer, 1,1'dioctadecyl-3,3,3'3'-tetramethyleindocarbocyanine perchlorate (DiI) was injected into the PMM, and combined with serotonin, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and phenyl N-methyltransferse (PNMT) immunocytochemistry to reveal neuroanatomical connections. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridisation (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. Cells labelled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA-MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labelled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin/catecholamine neuronal distribution and their variable interactions with PMM DA-MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality.

Our previous studies using turkey hens have demonstrated that c-fos mRNA (a marker of neuronal activation) is expressed in gonadotrophin-releasing hormone-I (GnRH-I), vasoactive intestinal peptide (VIP) and dopamine (DA) neurones following electrical stimulation in the preoptic area. DA has been shown to have both stimulatory and inhibitory effects on the GnRH-I/luteinising hormone (LH), follicle-stimulating hormone (FSH) and VIP/prolactin (PRL) systems. To identify the DA neurones that mediate the stimulatory influences of photoperiod on the reproductive system, we examined c-fos mRNA induction in DA, GnRH-I, and VIP neurones in the turkey hypothalamus using a dark-interruption experimental design. A 30-min light period was provided to short day (6L : 18D) photosensitive turkeys at times when birds were responsive to light (14 h after first light) and at times when birds were unresponsive to light (8 h and 20 h after first light). The only area where DA neurones were activated when the birds were provided with light was in the nucleus premammillaris (PMM). The number of activated DA neurones was significantly greater when light was provided at 14 h (during the photoinducible phase) than at 8 h or 20 h. At 14 h, there was also an increase in the number of GnRH-I neurones activated in the area of the nucleus commissura pallii (nCPa), as well as an up-regulation of GnRH-I mRNA expression. No expression of c-fos mRNA was observed in VIP neurones in the nucleus infundibularis or up-regulation of VIP mRNA expression in any of the experimental light treatments. These results are the first evidence to demonstrate a relationship between the dopaminergic system in the PMM and the GnRH-I system in the nCPa during the photoinduction of avian reproductive activity.

Our recent work has demonstrated that dopamine, acting through D 2 dopamine receptors on pituitary cells, inhibits the stimulatory effects of vasoactive intestinal peptide (VIP) on prolactin release and prolactin gene transcription. It is hypothesised that the stimulatory and inhibitory roles of VIP and dopamine, respectively, on prolactin synthesis and release are mediated by their opposite effects on intracellular Ca 2+ concentration ([Ca 2+ ] I ) in lactotrophs. The present study aimed: (i) to investigate the effect of VIP and dopamine on [Ca 2+ ] I of cultured turkey anterior pituitary cells and (ii) to examine the role of Ca 2+ signalling in mediating the regulatory effects of VIP and dopamine on prolactin mRNA levels and prolactin release. Changes in [Ca 2+ ] I were measured spectrofluorometrically using Fura-2/AM as a fluorescent Ca 2+ indicator. Semi-quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to determine prolactin mRNA levels and prolactin release, respectively. VIP or the L-type Ca 2+ channel activator, Bay K8644 (Bay) increased [Ca 2+ ] I in a concentration- and time-dependent fashion, an effect abolished by preincubating the cells with R(-)-propylnorapomorphine HCl, a D 2 dopamine receptor agonist (D 2 AG) or Verapamil (VR), a specific L-type Ca 2+ channel blocker. Similarly, either VR or D 2 Ag diminished the VIP/Bay stimulatory effect on prolactin expression and release. On the other hand, pretreatment of pituitary cells with thapsigargin (TG) or neomycin (NEO), to deplete the intracellular Ca 2+ stores, showed no effect on basal or VIP-stimulated prolactin mRNA levels; although VIP- induced prolactin release was partially inhibited by NEO but not TG. These results suggest that intracellular Ca 2+ represents a common signal transduction pathway through which VIP and dopamine can exert antagonistic control on prolactin synthesis and release in avian lactotrophs.

Leptin is a 16 kDa protein hormone that besides being a satiety factor also functions as a pleiotropic molecule regulating endocrine and immune functions. The aim of this study was to investigate the role of leptin on mitogen stimulated T-lymphocyte proliferation in birds. In the first experiment (in vitro), peripheral blood was collected from turkeys and lymphocytes were isolated from samples. Recombinant chicken leptin was added at concentrations of 0, 10 −9 , 10 −8 , 10 −7 , and 10 −6 M prior to mitogen stimulation with Concavalin A. BrdU incorporation allowed us to detect T-cell proliferation using intracellular labeling of nucleic acids. Mitogen activation induced with Concavalin A caused a proliferation of T-cells that was positively correlated with the concentration of leptin. In the second experiment (in vivo), asian blue quail were fitted with osmotic pumps releasing leptin and injected with phytohemagglutinin (PHA) in their wing-webs before, during, and after leptin administration. The response to mitogen was greater in leptin treated birds during the leptin administration, but not before or after. These findings demonstrate that leptin enhances mitogen stimulated T-cell proliferation in birds. The results correspond with previous reports on mammals.

Reproductive failure associated with heat stress is a wellknown phenomenon in avian species. Increased prolactin (PRL) levels in response to heat stress have been suggested as a mechanism involved in this reproductive malfunction. To test this hypothesis, laying female turkeys were subjected to 408C for 12 h during the photo-phase daily or maintained at 24‐268C. Birds in each group received oral treatment with parachlorophenyalanine (PCPA; 50 mg/kg BW/day for 3 days), an inhibitor of serotonin (5-HT) biosynthesis, or immunized against vasoactive intestinal peptide (VIP). Both treatments are known to reduce circulating PRL levels. Nontreated birds were included as controls. In the control group, high ambient temperature terminated egg laying, induced ovarian regression, reduced plasma luteinizing hormone (LH) and ovarian steroids (progesterone, testosterone, estradiol) levels, and increased plasma PRL levels and the incidence of incubation behavior. Pretreatment with PCPA reduced (P , 0.05) heat stress-induced decline in egg production, increase in PRL levels, and expression of incubation behavior. Plasma LH and ovarian steroid levels of heat stressed birds were restored to that of controls by PCPA treatment. As in PCPA-treated birds, VIP immunoneutralization of heat-stressed turkeys reduced (P , 0.05) circulating PRL levels and prevented the expression of incubation behavior. But it did not restore the decline in LH, ovarian steroids, and egg production (P . 0.05). The present findings indicate that the detrimental effect of high temperature on reproductive performance may not be related to the elevated PRL levels in heat-stressed birds but to mechanism(s) that involve 5-HT neurotransmission and the induction of hyperthermia. birds, egg production, heat stress, incubation behavior, luteinizing hormone, ovary, pituitary hormones, prolactin, reproduction, reproductive hormones, serotonin, stress, turkeys, vasoactive intestinal peptide

Active immunization of turkey hens against vasoactive intestinal peptide (VIP) has been shown to inhibit incubation behavior and to increase egg production in second-cycle hens. The objective of this study was to compare the effect of VIP immunization on first- and second-cycle turkey hens during a 27-wk production period. First- (25-wk-old) and second- (54-wk-old) cycle hens were intermixed, distributed among 16 pens, and subjected to a photoperiod of 6 h of light and 18 h of darkness for 10 wk. The first-cycle hens were divided into two groups: keyhole limpet hemocyanin (KLH)-immunized controls (n = 16) and VIP-immunized (n = 18). Second-cycle hens were divided into four groups: 1) unimmunized controls (n = 19), 2) KLH-immunized controls (n = 18), 3) VIP-immunized (n = 19), and 4) VIP-preimmunized (immunized during first cycle; n = 16). Each hen received four antigen injections beginning the day of photostimulation (4-wk intervals), except for the preimmunized hens, which received three injections beginning 4 wk after photostimulation. The maximum titer of VIP antibodies in first-cycle, second-cycle, and preimmunized hens was 17.2+/-2.2, 20.9+/-2.9, and 21.7+/-3.2%, respectively. After photostimulation, plasma prolactin of first- and second-cycle control hens peaked between 484 +/-105 and 630+/-118 ng/mL. In contrast, prolactin changed very little in VIP-immunized turkeys. The average number of daily nest visits was less in first- and second-cycle VIP-immunized hens (1.68+/-0.23 and 1.09+/-0.15 visits per hen per day, respectively) than in their respective KLH-immunized controls (2.47+/-0.36 and 2.65+/-0.45 visits per hen per day). Expression of incubation behavior was 50.0 and 52.6% in first- and second-cycle control hens, respectively, upon termination of the study. In contrast, only 11.1% first-cycle and 5.2% second-cycle VIP-immunized turkeys exhibited the hormonal and behavioral characteristics of incubating hens. Average weekly egg production of first- and second-cycle VIP-immunized turkeys was similar (3.58+/-0.19 vs. 3.63+/-0.14 eggs per hen per wk). First- and second-cycle control hens laid 2.63+/-0.25 and 2.41+/-0.20 eggs per hen per wk, respectively. The present results show that comparable egg production was attained in first- and second-cycle hens by active immunization with VIP.

To characterize further vasoactive intestinal peptide (VIP) as the prolactin-releasing factor in avian species, the present study examined hypothalamic VIP transcription and plasma prolactin (PRL) levels during the turkey reproductive cycle. The contribution of transcription to hypothalamic VIP mRNA steady-state levels and VIP content in response to gonadal stimulating photoperiod was also investigated. Nuclear run-on transcription assays were performed using nuclei isolated from hypothalami. Cytoplasmic VIP mRNA levels, and VIP content in the median eminence and plasma PRL levels were determined by Northern blot analysis and radioimmunoassays respectively. The alterations in VIP transcription mirrored the changes in cytoplasmic VIP mRNA and VIP content during the reproductive stages. VIP transcription, cytoplasmic VIP mRNA level and VIP content were lowest in non-photostimulated birds, higher (P0.05) were not observed during the transition from incubation to photorefractoriness, even though there was a sharp decline in circulating plasma PRL levels (P

The objective of this study was to culture turkey hypothalami and examine vasoactive intestinal peptide (VIP) release during the turkey reproductive cycle. The release of VIP was studied employing a computer-guided perifusion system. Hypothalami were perifused with Krebs-Ringer bicarbonate medium for 10 or 15 h at a flow rate of 40 microliter/min, and perifusate was collected at 5-min intervals. Basal VIP secretion increased (p 0.05) in VIP release in response to DA stimulation between hypothalamic fragments obtained from nonphotostimulated and incubating birds. The data suggest that 1) a VIP pulse generator appears to be located within the turkey hypothalamus, on the basis of the observed pulsatile release of VIP; 2) hypothalamic secretion of VIP is augmented by removal of inhibitory factors residing outside of the hypothalamus, or by the loss of negative feedback mechanisms that inhibit VIP release; and 3) mechanisms responsible for altering VIP release during different reproductive conditions may lie external to the hypothalamus.

Wilson's Phalarope (Phalaropus tricolor) is a sex role-reversed species in which incubation of eggs and care of young is performed exclusively by the male. Plasma levels of prolactin (PRL), the hormone most associated with parental care in birds, are higher in incubating males than in nonincubating males or females. Conversely, plasma testosterone levels are reduced in males during incubation. In an attempt to characterize the physiological basis of this unusual parental care system we used quantitative film autoradiography and densitometry to measure the specific binding in vitro of 125I-ovine PRL to 12 brain regions in females, nonincubating males, and incubating males during the normal breeding season. We also measured hypothalamic chicken gonadotropin-releasing hormone I (cGnRH-I) in three brain areas in these same birds, as well as plasma levels of PRL and testosterone. Analysis revealed that cGnRH-I concentrations in the preoptic area and plasma testosterone levels were significantly lower in incubating males than in nonincubating males. Specific binding of 125I-ovine PRL was detected in choroid plexus and in several diencephalic brain regions of both sexes, with highest binding activity recorded in the dorsolateral thalamus, medial habenula, nucleus subrotundus, and preoptic area. When adjustments were made for the large number of comparisons performed, specific binding did not vary significantly by sex or breeding stage in any single brain region. However, average specific binding values in nonincubating males exceeded those of incubating males in 9 of the 11 PRL-sensitive regions examined. Increased occupancy of the receptor by endogenous PRL during incubation could have contributed to this result, since plasma PRL levels were elevated in incubating males. In addition, PRL binding activity in several of these brain regions tended to correlate negatively with plasma PRL. The two exceptions to this general pattern were the preoptic area and the lateral septum, where mean specific binding was 14-15% higher in incubating males than in nonincubating males. This raises the interesting possibility that PRL sensitivity is up-regulated during incubation in some regions of the male phalarope brain, such as the preoptic area and lateral septum, that have been implicated in PRL-modulated changes in behavior and reproductive activity during this breeding stage.

The present study examined turkey prolactin (PRL) transcription and PRL mRNA stability during different reproductive stages. Nuclear run-on transcription assays were performed using isolated nuclei from pituitaries of turkeys at different reproductive stages. Meanwhile, cytoplasmic PRL mRNA and plasma PRL were measured by slot blot and RIA respectively. The PRL transcription, pituitary cytoplasmic PRL mRNA abundance and plasma PRL levels increased after photostimulation and peaked at the incubating stage (PPPP>0·05). The half-life of PRL mRNA was determined in pituitaries of non-photostimulated, laying, incubating and photorefractory hens. Primary pituitary cell cultures were treated with the transcription inhibitor actinomycin-D and the decay of the pre-existing PRL mRNA was quantified using Northern blot analysis. The PRL mRNA half-life was 1·5- and 1·4-fold greater in incubating and laying birds respectively than in non-photostimulated turkeys (P

Vasoactive intestinal peptide (VIP) is a significant prolactin-releasing factor (PRF) in avian species, and dopamine (DA) exhibits both a stimulatory and inhibitory influence upon this prolactin (PRL) secretion. The stimulatory effect of DA upon PRL release appears to be mediated by VIP. This study investigated DAergic actions upon VIP release using turkey hypothalamic explants perifused with DA and its agonists or antagonists. VIP release was stimulated by DA in a dose-dependent manner (10 nmol DA/min, from 67.2 +/- 3.9 to 164.3 +/- 3.1 pg/5 min; 100 nmol DA/min, from 70.1 +/- 3.2 to 291.0 +/- 7.5 pg/5 min; 1,000 nmol DA/min, from 72.0 +/- 4.8 to 501.0 +/- 24.7 pg/5 min). The D1 DA receptor antagonist (R+)-SCH-23390 HCl completely negated the stimulatory effect of DA (100 nmol/min) upon VIP release. Perifusion with the D2 DA receptor antagonist S(-)-eticlopride HCl by itself stimulated VIP release from the hypothalamic explants, increasing VIP from 38.1 +/- 5.3 to 161.9 +/- 9.7 pg/5 min, where release stabilized until perifusion was terminated. The D1 DA agonist (+)-SKF-38393 HCl increased VIP release from 52.7 +/- 4.6 to 192.6 +/- 16.9 pg/5 min, and this stimulated release was partially inhibited by the D2 DA receptor agonist R(-)-NPA HCl (from 192.6 +/- 16.9 to 139.7 +/- 13.8 pg/5 min). These results suggest that VIP secretion is in part regulated by possible opposite actions between stimulatory D1 and inhibitory D2 DA receptors in the turkey hypothalamus.

Photostimulation initiates and maintains the rise in circulating prolactin (PRL) observed during the reproductive cycle of the female turkey. Vasoactive intestinal peptide (VIP) is the principal PRL-releasing factor. This study tested the hypothesis that gonadal stimulatory photoperiods stimulate PRL secretion by releasing hypothalamic VIP. Therefore, an experiment was designed to determine if VIP immunoneutralization altered photo-induced PRL secretion. Reproductively quiescent female turkeys were divided into two groups comprising turkeys actively immunized with synthetic VIP conjugated to keyhole limpet hemocyanin (VIP-KLH; immunized; n = 48) or KLH alone (control; n = 48). The first immunization was administered 6 weeks before photostimulation. Blood samples were collected at frequent intervals prior to and following photostimulation, and plasma PRL concentrations were determined. Vasoactive intestinal peptide antibody titer was estimated from the percentage of 125I-labeled VIP bound to plasma diluted 1:1000. At the onset of photostimulation (Day 0), plasma PRL levels were similar for immunized and control turkeys (9.1 +/- 0.3 versus 8.9 +/- 0.3 ng/ml, respectively). Plasma PRL of control birds increased (P < 0.05) by Day 16 of photostimulation, reaching a peak value of 724.9 +/- 90.1 ng/ml on Day 84. In contrast, plasma PRL remained essentially unchanged in immunized birds. Titer of anti-VIP antibodies expressed as 125I-VIP bound by plasma in immunized birds was 10.9 +/- 1.5% on the day of photostimulation. Incubation behavior was blocked in immunized birds, whereas 75% of controls exhibited incubation behavior. The control group laid 1.83 eggs/ week/hen compared to 3.40 eggs/week/hen in immunized hens. These findings suggest that photoperiodic modulation of PRL secretion in the turkey is influenced by hypothalamic VIP neuronal system.

It is well documented that vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor and that dopamine (DA) is an inhibitory neurotransmitter in avian species. However, the roles of VIP and DA in the regulation of PRL gene expression are unclear. In this study, primary anterior pituitary cells cultured from laying turkeys were utilized to investigate the influence of VIP and dopaminergic D1 and D2 receptors on PRL secretion, PRL mRNA, and PRL synthesis. Incubation of pituitary cells with VIP increased PRL secretion up to 3.5-fold within 3 hr. Prolactin mRNA was undetectable during the first 2 hr of pituitary cell treatment; thereafter, the PRL mRNA content response to VIP increased with 24-48 h (P < 0.05). Total PRL content (media + cellular) increased over time in the presence of VIP. The response of cells incubated in the presence of a dopaminergic D1 receptor agonist (SKF38393) was variable and inconclusive. However, cells incubated with a dopaminergic D2 receptor agonist (quinpirole) inhibited VIP-induced PRL secretion (P < 0.05) and PRL mRNA levels (P < 0.05) in a dose-related fashion without effect on the basal levels of PRL release and PRL mRNA. These observations suggest that VIP, in addition to acting as a PRL-releasing peptide, also plays a role in the regulation of PRL gene expression. Moreover, the results of this study also indicate that a drug that can selectively stimulate dopamine D2 receptors can also regulate PRL secretion and PRL mRNA in turkey pituitary cells in culture.

The purpose of this study was to examine the role of vasoactive intestinal peptide (VIP) in prolactin (PRL) release in young turkey poults. Poults obtained from hens immunized with keyhole limpet hemocyanin (KLH)-chicken VIP (cVIP-KLH) conjugate or with KLH alone were used in this study. Plasma VIP antibody was tested by means of monoiodinated cVIP. Plasma samples were prediluted (1:100) in 0.05 EDTA PBS and used as a source of primary antibody in a cVIP-binding assay. Antibody levels averaged 50.8 +/- 10.3% at hatch and then declined with age to a level of 5.0 +/- 2.1% after 3 wk. Plasma PRL concentration was lower in cVIP-KLH-immunized poults (p < 0.05) than in KLH-immunized birds. Exogenous VIP administration at 7 days of age (7.8 micrograms/kg) increased plasma PRL level (p < 0.05) to a peak value of 359 +/- 32 ng/ml in the KLH-immunized birds. A smaller increase (p < 0.05) was obtained when the KLH-immunized poults received injections at 2 and 3 wk of age. The PRL response to cVIP administration was not observed in 1- and 2-wk-old poults maternally immunized with cVIP-KLH. Similarly, electrical stimulation of the ventromedial nucleus of the hypothalamus, at 1 and 8 days of age, induced a significant increase in the plasma PRL level of maternally KLH-immunized birds but not in maternally cVIP-KLH-immunized birds. These findings suggest that cVIP is a PRL-releasing factor in young turkey poults, similar to the finding in adult turkey hens.

Cockatiel eggs hatch asynchronously because most eggs are laid after continuous incubation begins. We manipulated numbers of eggs in the nest after cockatiels began incubating to determine whether number of eggs in the nest influenced clutch size. We also measured plasma prolactin 2, 7, 12, and 17 days after egg laying began to determine whether clutch size covaried with prolactin concentration. We found that total egg production was stimulated in pairs for which the rate of egg accrual was reduced by egg removal, while total egg production was inhibited in pairs for which the egg accrual rate was increased by egg addition. Female, but not male, plasma prolactin concentrations correlated negatively with egg production on day of lay 17 but not before. We conclude that egg production in incubating cockatiels is influenced by feedback from the number of eggs in the nest and that clutch termination may be signaled by rising prolactin levels in females.

Although vasoactive intestinal peptide (VIP) is a well characterized physiological PRL-releasing factor in avian species, its regulated expression is not fully understood. We cloned complementary DNAs encoding the prepro-turkey VIP (prepro-tVIP) molecule from an adult turkey hypothalamic complementary DNA library. When the amino acid sequence of the prepro-tVIP was compared to chicken and mammalian sequence, it was found that the isolated tVIP molecules lacked the 27-amino acid peptide histidine isoleucine (PHI) portion of the precursor protein. Several tissues showed an alternatively spliced tVIP transcript that lacked the PHI sequence. Only in the hypothalamus did tVIP-specific primer pairs and reverse transcription-polymerase chain reaction produce two alternatively spliced fragments. The larger hypothalamus-specific fragment was subjected to nucleotide sequence analysis and identified as containing the alternatively spliced PHI-containing exon, which encoded a 27-amino acid PHI peptide in addition to the 8 amino acids that flanked the peptide. Hypothalamic tVIP expression was shown to be up-regulated during the incubation phase of the reproductive cycle. The increased steady state level of tVIP messenger RNA appears to be regulated by nesting behavior, because nest deprivation dramatically suppressed its expression. Levels of the minor tVIP transcript containing both the PHI- and VIP-encoding exons did not significantly change between reproductive stages and were maintained at approximately 4-6% of the total tVIP transcript level. Our findings provide further evidence that VIP is the most important PRL-releasing factor in birds. Our study should serve as a useful model for determining whether PHI contributes in any way to the physiological role of PRL regulation. Revealing the tissue distribution of VIP and PHI gene expression and tissue-specific alternative splicing could contribute to an understanding of the physiological functions of the two peptides as well as their relative roles in PRL regulation.

Dopamine (DA) was infused into the third ventricle of anesthetized laying turkey hens at various concentrations to determine its effect on both basal prolactin (PRL) levels and ongoing electrically induced PRL secretion. The infusion of DA at rates of 1.0 or 10.0 nmol/min resulted in dose dependent increases in plasma PRL. These infusions had no inhibitory effect on electrically stimulated PRL release. The infusion of DA at 100.0 or 500.0 nmol/min caused no stimulation of PRL secretion and totally inhibited the PRL response elicited by electrical stimulation of the medial preoptic nucleus. These results show that dopaminergic influences are involved in both stimulating and inhibiting avian PRL secretion and suggest possible biphasic actions of DA within the brain.

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.

Vasoactive intestinal peptide (VIP) is a hypothalamic prolactin (PRL)-releasing factor in the turkey. The hypothesis in the present study was that active immunization of turkeys with VIP would neutralize endogenous VIP, decrease circulating PRL, and consequently prevent the expression of incubation behavior. Large white female turkeys were divided into three experimental groups comprising untreated controls, control turkeys immunized with keyhole limpet hemocyanin (KLH), and turkeys immunized with synthetic chicken VIP conjugate (KLH-cVIP). Each turkey received four injections at 4-wk intervals, starting on the day of photostimulation. The immune response, measured by the percentage binding of monoiodinated chicken VIP (cVIP) to plasma at a dilution of 1:1000, averaged 11.8 ± 2.5% during the reproductive life cycle. Immunization against KLH-cVIP prevented the normal increases of PRL that are associated with the photo-induced reproductive cycle. Over a 21-wk period beginning at photostimulation, KLH-cVIP-immunized birds exhibited a maximal plasma PRL level of 82.2 + 29.5 ng/ml, compared to 367.7 + 66.6 ng/ml and 227.5 + 51.7 ng/ml for non- and KLH-immunized turkeys, respectively. The total number of nest visits per hen during the 147-day experimental period decreased from 320.0 ± 48.2 in the nonimmunized controls to 180.7 53.7 and 149.4 ± 13.1 visits in KLH- and KLH-cVIP conjugate-immunized turkeys. Turkeys that showed an immune response to KLH-cVIP immunization did not exhibit incubation behavior, whereas 54% and 33% of non- and KLH-immunized hens incubated their eggs. The latter two groups laid 58.4 ± 7.2 eggs and 65.6 ± 9.9 eggs per hen, respectively, compared to 87.6 ± 3.1 eggs per hen for the KLH-cVIP-immunized turkeys. It is concluded that the increased egg production observed in KLH-cVIP-immunized turkeys is the result of failure to express incubation behavior due to lower PRL levels.

This study was designed to examine changes in cytochrome P450 17a-hydroxylase (C17) and aromatase (ARO) mRNA contents in the theca layer of preovulatory follicles (POF) as turkey hens transit from egg laying to incubation. Hens were grouped into the following categories: 1) laying hens-laid one egg per day and nested 1-2 times per day; 2) transitional hens-laid one egg per day and nested > 4 times per day; and 3) Day 1, Day 3, and Day 5 incubating hens-laid no eggs for 2, 4, or 6 days, respectively, and nested > 4 times per day. Small white follicles (SWF) and the theca layer from the largest (F.) and the third (F3), fifth (F 5), and seventh (F 7 ) largest POF were dispersed and challenged with testosterone (T) for 5 h. Relative levels of C17 and ARO mRNA were examined from the theca layers of F 1, F3, F, F 7, and SWF. The number of atretic follicles increased from O (layers) to 8 (Day 5 incubating hens). Serum LH, progesterone (P), and estradiol (E), but not T, declined on Day 1 of incubation. Basal levels of P, T, and E from theca and SWF cells declined in incubating hens. Both basal and T-stimulated theca and SWF production of E decreased in incubating hens. C17 and ARO mRNA declined in SWF, F 7, and F5 during follicular atresia. It is suggested that reduced gene expression of ovarian steroidogenic enzymes may be a partial determinant of reduced circulating sex steroid levels in incubating hens. Follicular maturation is associated with a progressive decrease in C17 and ARO mRNA steady state levels as the follicle matures; this may partially explain the decreases in T and E secretion that are associated with follicular maturation.

Hyperprolactinemia is associated with incubation behavior in avian species. Increased nesting activity is a major indication of incubation behavior. Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) secretion from the anterior pituitary. The goal of this study was to induce incubation behavior by stimulating PRL through chronically infusing VIP into the third ventricle of turkey brains. In experiment 1, porcine VIP (pVIP) was infused into the median eminence at a rate of 60 ng/min for 7 days by means of osmotic pumps implanted s.c.. Plasma PRL increased significantly in the pVIP-treated turkeys (p < 0.001). Although egg laying was not affected by the pVIP infusion, the mean oviduct weight decreased (p < 0.057). In experiment 2, saline or pVIP (30 or 60 ng/min) was infused into the third ventricle of laying turkeys for 12 days. Both pVIP treatments increased plasma PRL for 9 days (p < 0.05). The 30-ng pVIP/min infusion decreased nesting activity, plasma LH, ovary and oviduct weight, hypothalamic GnRH I, and anterior pituitary VIP receptors (p < 0.1). However, ovine PRL infusion (20.8 ng/min) into the same turkey flock increased nesting activity (p < 0.01). In conclusion, pVIP does not induce incubation behavior in laying turkeys.

We investigated the neuroendocrine changes involved in the transition from incubating eggs to brooding of the young in turkeys. Numbers of mesotocin (MT; the avian analog of mammalian oxytocin) immunoreactive (ir) neurons were higher in the nucleus paraventricularis magnocellularis (PVN) and nucleus supraopticus, pars ventralis (SOv) of late stage incubating hens compared to the layers. When incubating and laying hens were presented with poults, all incubating hens displayed brooding behavior. c-fos mRNA expression was found in several brain areas in brooding hens. The majority of c-fos mRNA expression by MT-ir neurons was observed in the PVN and SOv while the majority of c-fos mRNA expression in dopaminergic (DAergic) neurons was observed in the ventral part of the nucleus preopticus medialis (POM). Following intracerebroventricular injection of DA or oxytocin (OT) receptor antagonists, hens incubating eggs were introduced to poults. Over 80% of those injected with vehicle or the D1 DA receptor antagonist brooded poults, while over 80% of those receiving the D2 DA receptor antagonist or the OT receptor antagonist failed to brood the poults. The D2 DA/OT antagonist groups also displayed less c-fos mRNA in the dorsal part of POM and the medial part of the bed nucleus of the stria terminalis (BSTM) areas than did the D1 DA/vehicle groups. These data indicate that numerous brain areas are activated when incubating hens initially transition to poult brooding behavior. They also indicate that DAergic, through its D2 receptor, and MTergic systems may play a role in regulating brooding behaviors in birds.

An inverse relationship often exists between prolactin (Prl) and LH in avian species. Our study was designed to investigate the relationship between hypothalamic and posterior pituitary vasoactive intestinal peptide (VIP)--an endogenous Prl-releasing peptide--and chicken GnRH-I and GnRH-II. Hypothalamic VIP content was increased after photostimulation, reaching its highest levels in incubating and photorefractory birds. The highest hypothalamic GnRH-I content was in laying hens followed by that in photostimulated and incubating birds. The lowest levels were in the nonphotostimulated birds. Hypothalamic GnRH-II increased after photostimulation, then fell to nonphotostimulated levels during incubation and photorefractoriness. Posterior pituitary VIP content was elevated in response to photostimulation, reaching a peak value in the laying and incubating birds, then declining in the photorefractory hens. Posterior pituitary GnRH-I and GnRH-II content peaked in the incubating birds. Ovariectomy caused a significant reduction in hypothalamic GnRH-I and GnRH-II with no effect on VIP; no changes were found in the posterior pituitary due to ovariectomy. Reducing day length caused a significant decrease in the hypothalamic and the posterior pituitary content of VIP and GnRH-I, and GnRH-II. Ovine Prl (oPrl) administration to laying hens reduced the hypothalamic VIP and GnRH-I and GnRh-II content. Posterior pituitary GnRH-I content was also reduced. Although GnRH-II levels were reduced by Day 4 of injections, they rose to peak levels by Day 14 of oPrl administration. Posterior pituitary VIP content was not altered by oPrl.

We have investigated the effects of ovariectomy (OVX) and photostimulation (PS) on anterior pituitary LH beta mRNA abundance and circulating levels of LH in the domestic turkey. The birds were divided into the following four treatment groups: non-PS SHAM, PS SHAM, non-PS OVX, and PS OVX. Photostimulation was initiated 7 days after OVX. Anterior pituitaries and blood samples were collected on Days 0, 1, 3, 7, 14, and 26 after PS. The experiment was terminated when the PS SHAM birds were laying eggs regularly. Photostimulation of intact birds induced an increase in both LH beta mRNA levels (Days 3, 14, and 26 of PS) and serum LH (Days 14 and 26 of PS; p 0.05). Our results indicate that OVX and PS appear to be additive in stimulating LH secretion but do not have this effect on LH beta mRNA levels. We have hypothesized that gonadectomy induces near-maximal accumulation of LH beta mRNA and that therefore the subsequent stimulus of a long photoperiod has no additional effect. We suggest that increases in serum LH levels, beyond those of the OVX bird, may be controlled post-transcriptionally.

The effect of ovine prolactin (oPRL) administration to laying and incubating turkey hens was studied. In experiment one, five treatment groups (n = 6) of laying hens received injections of vehicle or oPRL (4 mg/bird/day) for 2, 4, 8, or 14 days. Hypothalamic vasoactive intestinal peptide (VIP) content decreased to its lowest level (p < 0.05) by Day 2 of oPRL injection. Serum turkey PRL decreased by Day 8 of injection, reaching nadir by Day 14. Similar to the decline in hypothalamic VIP content, the maximal decline in the number of anterior pituitary VIP binding sites was achieved by Day 2 of oPRL administration. Hypothalamic GnRH-I content decreased after 2 days of oPRL injection and remained low through Day 14 of the experiment. GnRH-II levels declined with time, reaching significantly lower levels after 8 days of injections, and remained low through Day 14. Plasma LH levels also declined (p < 0.05) after Day 14 of oPRL administration. In experiment 2, two groups (n = 6) of incubating birds were used: controls receiving injections of vehicle only and birds receiving injections of oPRL (4 mg/bird/day) for 10 days. Exogenous oPRL had no effect on hypothalamic VIP, GnRH-I or II, anterior pituitary VIP binding sites, or plasma turkey PRL or LH. The findings suggest that in laying hens, PRL inhibits its own secretion by acting on both the hypothalamus and the anterior pituitary; this phenomenon does not occur in incubating birds. Furthermore, we provide evidence that pRL acts centrally to reduce LH levels by reducing GnRH levels in the hypothalamus.

Vasoactive intestinal peptide (VIP) is a prolactin-releasing factor in turkey hens. Membranes from anterior pituitaries of turkey hens were used to characterize VIP receptors. Using HPLC-purified monoiodinated VIP, we found specific VIP receptors in the anterior pituitary glands. Binding was saturable and was time- and temperature-dependent. Scatchard analysis of competitive binding studies indicated two binding sites, a high-affinity binding site (Kd1) of 6.6 +/- 1.8 pM and maximum binding (Bmax1) of 1.52 +/- 0.2 pM, and a low-affinity binding site (Kd2) of 542 +/- 200 pM and Bmax2 of 15.8 +/- 8.0 pM. Binding of VIp to pituitary membranes was specific, as compared to other peptides of the glucagon family. The rank order of potency of the peptides tested was chicken VIP > porcine VIP > peptide histidine isoleucine > secretin > glucagon > growth hormone-releasing factor. Two binding sites were found in all the examined reproductive stages. The lowest binding site levels were found in nonphotostimulated and photorefractory birds, followed by photostimulated birds and layers; highest levels were found in incubating birds. Nest deprivation significantly reduced Bmax1 levels without changing hypothalamic VIP content. These results suggest the involvement of the anterior pituitary VIP receptors in the regulation of prolactin secretion from the pituitary gland.

As a consequence of its fundamental role in avian reproduction, incubation behavior has been of great interest to scientists and to turkey breeders and producers of hatching eggs. Incubation behavior in turkey hens continues to be a major hindrance to enhanced reproductive performance in the domestic turkey, resulting in substantial loss of potential egg production. The onset and maintenance of incubation behavior is dependent upon a series of obligatory factors. Nest entry induced by progesterone surges in laying hens, which is dependent on prior estrogen priming, is a prerequisite for prolactin (Prl)-induction of incubation behavior. Thereafter, persistent nesting stimulates Prl secretion and elevated Prl levels maintain incubation activity. The increased Prl secretion in incubating hens is related to a diminished dopaminergic and an increased vasoactive intestinal peptidergic activities in the hypothalamus. An increase in Prl gene expression and Prl releasing capacity of the pituitary may also contribute to the hyperprolactenimic state in the incubating bird.

A series of experiments were conducted to elucidate the role of the ovary in incubation behavior-associated luteinizing hormone (LH) suppression. Ovariectomy (Ovx) increased (p 0.05). Ovx had no effect on intramuscularly injected mammalian luteinizing hormone-releasing hormone (4 µg/kg i.m. > induced LH in incubating hens, but enhanced the LH response in laying hens. Serum LH and prolactin (Prl) were unaffected by Ovx, and nest deprivation (ND) decreased Prl levels (p

The relationships of prolactin (PRL) and LH messenger (m) RNA to serum and pituitary content were determined for turkey hens at different phases of the reproductive cycle. In the nonphotostimulated, reproductively inactive hen, serum and pituitary PRL content and pituitary PRL mRNA levels were low. All three PRL values rose after photostimulation and peaked during the incubation phase. Relative to nonphotostimulated hens, hyperprolactinemic incubating hens showed 220-, 11-, and 57-fold increases in serum PRL, pituitary PRL content, and pituitary PRL mRNA levels, respectively. These peak levels declined 80-, 3-, and 6-fold, respectively, in photorefractory hens. In contrast to PRL levels, serum LH, pituitary LH, and pituitary LH beta-subunit mRNA levels did not change as dramatically. Serum LH showed no significant changes for the different reproductive phases. Pituitary LH peaked after photostimulation and declined to its lowest level in incubating hens. Pituitary LH-beta mRNA abundance was highest in photostimulated and laying hens and lowest in incubating and photorefractory hens. These results demonstrate that the abundance of LH-beta and PRL mRNA shows an inverse relationship in photostimulated/laying and incubating turkey hens.

In the turkey, the onset of incubation behavior is associated with altered ovarian steroidogenesis,ovarian regression, decreased LH secretion, and increased serum prolactin(Prl) levels.To clarifythe relativecontribution of circulating LH and PrI to the initiationof ovarian regression, laying hens were exposed for0, 3, 7, or 14 days to a forced molting procedure (exposure to reduced day length of 6L: 18D and removal of feed and water forthe initial 3 days) that induces ovarian regression and decreased LII levelsbut does not increase Pr! levels.On each of these days,hens were killed and granulosa and theca interna cells from the largest(Fl) and fifthlargest(F5) preovulatory folliclesand totalcellsfrom the smallwhite follicles(SWF) were incubated for 5 h in the presence or absence of ovine LII (0LH; 0-1 000 ng/ml). Force-molted hens exhibited diminished levels of circulating LH, Pr!,progesterone (P), androgen (A), and estradiol (E) by Day 3 of treatment. Ovarian atresia began in Fl by the third day of treatment, and included Ft and F5 by the seventh day. No preovulatory follicleswere present on the fourteenth day.With both Fl and F5 granulosa cells, production of P in the presence of oLH was initially enhanced (Day 3) and laterabsent (Day 7). In contrast, production of A by F5 theca internscellsin the presence of 0LH was initially suppressed (Day 3) and then returned to pretreatment levels (Day 7). Production of E by SWF cells was depressed by Day 3, further reduced by Day 7, and returned to Day 3 levelsby Day 14 of treatment.Thus, two contrasting patterns of alteredLH responsiveness occur between Psecretingcellsand A- and E-secretingcells followinga reduction of circulatingLH levelsinduced by forced molting. These initial changes in responsiveness to LII occurred concomitantly with depressed serum LII levelsand in the absence of elevated concentrations of PrI. We conclude that an increase in serum Prl is not required for the induction of gonadal regression or the steroidogenic changes associated with follicular atresia

1. Embryonic injections of 0.3 mg/egg of tamoxifen (TAM), 0.2 mg/egg CI‐628 (both antioestrogens), 0.5 mg/egg (ATD (aromatisation inhibitor), or antibodies to oestradiol (E), all suppressed male copulatory activity (MCA) in young male chicks. 2. Embryonic injections with either flutamide (F, androgen antagonist) or high dose of antibodies to testosterone (T) only slightly suppressed MCA. 3. TAM had no effect on embryonic plasma LH levels, 24 and 48 h after injection. 4. It seems that at the embryonic stage oestradiol is required for the normal differentiation of MCA.

The performance of 1,312 male market turkeys (Large White, Nicholas strain) from 0 to 20 wk of age fed diets varying in feed form and energy level was measured under two stocking densities (.21 or .46 m2 per bird) and four lighting and temperature programs. The four diets were 1) corn and soybean meal with 1% supplemental fat, mash (CSM); 2) as 1, pelleted (CSP); 3) as Diet 1 but with 1, 2, 4, 6, and 8% supplemental fat during 0 to 4, 4 to 8, 8 to 12, 12 to 16, and 16 to 20 wk of age, respectively (CSF); and 4) as Diet 1 but with barely included at 0, 20, 35, 50, and 65% during the respective 4-wk age periods (CSB). The four light and temperature programs were 1) Environment A with intermittent light [4 [2 h light (L):4 h dark (D)]] in combination with cycling temperature at 7 and 21 C during light and dark photoperiod, respectively; Environment B with intermittent light, 21 C; Environment C with continuous light (18L:6D) and cycling temperature of 7 to 21 C; and Environment D with intermittent light, 7 C. Lighting and temperature programs started at 1 and 4 wk of age, respectively. Body weights at 20 wk of age decreased (P less than .05) with increasing temperature (13.86 versus 12.26 kg for Environments D and B, respectively) with cycling temperature intermediate (13.51 kg for Environment A). Intermittent light (P less than .05) improved BW and feed conversion by 3.4 and 2.0%, respectively, compared with continuous light. Rearing males at .21 m2 per bird versus .46 m2 per bird decreased weight (P less than .05) by 5.5%. Twenty-week BW of males fed the CSP (13.52 kg) and CSF (13.58 kg) diets were greater (P less than .05) than those fed CSM (12.90 kg) and CSB (12.69 kg) diets. Significant (P less than .05) interactions between diet, environment, and density were not detected for most performance characteristics. Environmental measurements indicated higher dust and ammonia levels in the warm environment (B). Isolates of aspergillus and incidence of airsacculitis at time of processing were greatest in Environment B.

Intracranial perfusion of ovine prolactin (oPrl) via osmotic pump in laying turkey hens caused a sudden onset in incubation behavior, defined as an increase in nest visits. The hens also displayed a gradual decrease in egg laying during the time they were receiving oPrl, another indicator of the onset of incubation. Circulating immunoreactive turkey Prl levels fell during the perfusion period, even though the hens were displaying persistent nesting activity and reduced egg laying. No effects on serum LH were noted. Perfusion of oPrl during the first 14 days of photostimulation delayed the onset of egg laying by several days. No effects on serum Prl or serum LH were noted. It is suggested that incubation behavior is facilitated by central levels of Prl.

The hypothesis that vasoactive intestinal peptide (VIP) functions as a hypothalamic prolactin (PRL)-releasing peptide in the turkey was tested by determining the effects of hypothalamic VIP immunoneutralization and pituitary VIP receptor blockade on hypothalamic extract (HE)-induced PRL secretion from dispersed anterior pituitaries. Incubation of cells with porcine VIP (pVIP; 0.5 or 10 nM) significantly stimulated PRL secretion. This effect was inhibited in a dose-related manner by 1-hr preincubation of pVIP with a VIP antisera (A/S; 1:500-1:50,000). Likewise, HE (0.3 equivalent)-stimulated PRL secretion was inhibited by preincubation with VIP A/S (P less than 0.0001). A 96-98% reduction in PRL secretion was obtained from cells cultured with HE, that was previously incubated with 1/500 dilution of antiserum. Pretreatment of pituitary cells for 15 min with [4Cl-D-Phe6,Leu17] VIP, a VIP receptor antagonist (10(-5) M), significantly depressed the PRL response to 0.5 nM VIP (9.9 +/- 0.5 micrograms/500,000 cells vs 4.9 +/- 0.1 micrograms/500,000 cells; 22.4 +/- 0.9 micrograms/500,000 cells vs 14.7 +/- 0.4 micrograms/500,000 cells) or 0.3 eq HE (8.8 +/- 0.6 micrograms/500,000 cells vs 5.2 +/- 0.2 micrograms/500,000 cells; 15.3 +/- 0.3 micrograms/500,000 cells vs 8.2 +/- 0.2 micrograms/500,000 cells). These results suggest that hypothalamic stimulation of PRL secretion appears to be mediated by receptors specific for VIP and that VIP is an endogenous hypothalamic PRL-releasing peptide in the turkey.

Day length (photoperiod) is a powerful synchroniser of seasonal changes in the reproductive neuroendocrine activity in temperate-zone birds. When exposed to light during the photoinducible phase, reproductive neuroendocrine responses occur. However, the neuroendocrine systems involved in avian reproduction are poorly understood. We investigated the effect of light exposure at different circadian times upon the hypothalamus and components of the circadian system, using c-fos mRNA expression, measured by in situ hybridisation, as an indicator of light-induced neuronal activity. Levels of c-fos mRNA in these areas were compared after turkey hens (on a daily 6-h light period) had been exposed to a 30-min period of light occurring at 8, 14, or 20 h after the onset of first light of the day (subjective dawn). Non-photostimulated control birds were harvested at the same times. In birds, photostimulated within the photoinducibile phase (14 h), in contrast to before or after, c-fos mRNA was significantly increased in the nucleus commissurae pallii (nCPa), nucleus premamillaris (PMM), eminentia mediana (ME), and organum vasculosum lamina terminalis (OVLT). Photostimulation increased c-fos mRNA expression in the pineal gland, nucleus suprachiasmaticus, pars visualis (vSCN) and nucleus inferioris hypothalami compared to that of their corresponding nonphotostimulated controls. However, the magnitudes of the responses in these areas were similar irrespective of where in the dark period the pulses occurred. No c-fos mRNA was induced in the nucleus infundibulari, in response to the 30-min light period at any of the circadian times tested. The lack of c-fos up-regulation in the pineal gland and vSCN following photostimulation during the photoinducible phase lends credence to the hypothesis that these areas are not involved in the photic initiation of avian reproduction. On the other hand, c-fos mRNA increases in the nCPa, ME, and OVLT support other studies showing that these areas are involved in the onset of reproductive behaviour initiated by long day lengths. The present study provides novel data showing that the PMM in the caudal hypothalamus is involved in the neuronally mediated, light-induced initiation of reproductive activity in the turkey hen.

Day length cues are used by temperate zone birds to time seasonal changes in reproductive physiology and behavior. However, the neuronal and neurochemical circuits used to measure day length (photoperiodic time measurement; PTM), transduce light information and activate the reproductive neuroendocrine system have not been definitely established. Recent findings from our laboratory provide data showing dopamine (DA) neurons within the premammillary nucleus (PMM) of the caudal turkey hypothalamus are putative photoreceptive neurons. These neurons reach threshold activation when a brief pulse of light is provided during the photo-inducible phase for photosexual stimulation. To further clarify the role of PMM neurons in coding daylight information, we showed that by using double-label immunocytochemistry (ICC) these neurons are immunoreactive (ir) to both tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and melatonin (MEL). Moreover, we found these neurons to express tryptophan hydroxylase 1 (TPH1; the first enzyme in MEL biosynthesis) and 5-HT N-acetyltransferase (AANAT; a key regulatory enzyme in MEL synthesis) mRNAs but not neuronal tryptophan hydroxylase 2 mRNA (TPH 2; the rate limiting enzyme in 5-HT pathway). Both TH and TPH1 mRNAs were shown to cycle rhythmically, and with opposite phases, in PMM neurons of birds kept under a diurnal illumination cycle (12-h light/dark; LD). These neurons could also generate 24 h TH and TPH1 mRNA expression rhythms with the same phase relationship in constant light (LL) and constant dark (DD). In addition, the expression patterns and amplitudes of TH and TPH1 mRNAs were different between long and short photoperiods. These findings may form the basis for an endogenous dual-oscillator circadian system within PMM DA-MEL co-localized neurons controlling reproductive seasonality in birds.

The neural and neurochemical substrates regulating reproduction in birds remain vaguely defined. The findings that electrical stimulation in the medial preoptic area (ES/MPOA) or intracerebroventricular infusion of dopamine (DA) stimulated luteinising hormone (LH) and prolactin (PRL) release in female turkeys, led to the suggestion that ES/MPOA might help to clarify the DA circuitry regulating LH and PRL. We used c-fos mRNA and tyrosine hydroxylase immunoreactivity as measured by double in situ hybridisation/immunocytochemistry (ISH/ICC) to determine which group/subgroup of DA neurones was activated following unilateral ES/MPOA. To establish that the reproductive neuroendocrine system was activated, double ISH/ICC was also conducted on c-fos/gonadotrophin-releasing hormone-I (GnRH-I) and c-fos/vasoactive intestinal peptide (VIP). Changes in circulating LH and PRL were determined by radioimmunoassay. Unilateral ES/MPOA (100 microA, right side) of anaesthetised laying turkeys for 30 min increased circulating LH and PRL levels. It also induced c-fos mRNA expression on the ipsilateral side by all GnRH-I neurones within the septopreoptic region, implying that GnRH-I neurones in this region share similar circuitry. VIP neurones within the nucleus infundibularis were the only VIP group to show c-fos mRNA expression, suggesting their involvement in ES/MPOA induced PRL release. c-fos mRNA expression was also observed in a subgroup of DA neurones in the nucleus mamillaris lateralis (ML). To our knowledge, the present study is the first to show that activation of DAergic cells in the ML is associated with the activation of GnRH-I and VIP neurones and the release of LH and PRL. It is likely that ES/MPOA activated VIP/GnRH-I neurones via activation of DA neurones in the ML, as this was the only DA subgroup that showed c-fos mRNA expression.

Our recent work has demonstrated that dopamine, acting through D2 dopamine receptors on pituitary cells, inhibits the stimulatory effects of vasoactive intestinal peptide (VIP) on prolactin release and prolactin gene transcription. It is hypothesised that the stimulatory and inhibitory roles of VIP and dopamine, respectively, on prolactin synthesis and release are mediated by their opposite effects on intracellular Ca2+ concentration ([Ca2+]i) in lactotrophs. The present study aimed: (i) to investigate the effect of VIP and dopamine on [Ca2+]i of cultured turkey anterior pituitary cells and (ii) to examine the role of Ca2+ signalling in mediating the regulatory effects of VIP and dopamine on prolactin mRNA levels and prolactin release. Changes in [Ca2+]i were measured spectrofluorometrically using Fura-2/AM as a fluorescent Ca2+ indicator. Semi-quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to determine prolactin mRNA levels and prolactin release, respectively. VIP or the L-type Ca2+ channel activator, Bay K8644 (Bay) increased [Ca2+]i in a concentration- and time-dependent fashion, an effect abolished by preincubating the cells with R(-)-propylnorapomorphine HCl, a D2 dopamine receptor agonist (D2AG) or Verapamil (VR), a specific L-type Ca2+ channel blocker. Similarly, either VR or D2Ag diminished the VIP/Bay stimulatory effect on prolactin expression and release. On the other hand, pretreatment of pituitary cells with thapsigargin (TG) or neomycin (NEO), to deplete the intracellular Ca2+ stores, showed no effect on basal or VIP-stimulated prolactin mRNA levels; although VIP-induced prolactin release was partially inhibited by NEO but not TG. These results suggest that intracellular Ca2+ represents a common signal transduction pathway through which VIP and dopamine can exert antagonistic control on prolactin synthesis and release in avian lactotrophs.

1. Four methods of semen collecting that involved interruption of mating in two breeding ostrich pairs were tested: an artificial vagina was tested without promising results; the funnel method, in which a funnel was placed under the phallus of the tested male immediately after mating allowing semen drips to be collected; the vacuum method, using a turkey semen collector, inserted into the seminal canal; and the tube method, conducted by placing a test tube inside the seminal canal, allowing semen to enter by gravity. 2. For the funnel, vacuum and tube methods, respectively, average semen volume was 0.1 +/- 0.02, 1.12 +/- 0.22, and 0.58 +/- 0.13 ml, sperm concentration was 0.66 +/- 0.14, 2.35 +/- 0.26, and 2.13 +/- 0.27 x 10(9) cells/ml, and percentage of abnormal cells was 5.82 +/- 1.79%, 4.68 +/- 1.19%, and 7.09 +/- 1.72%. 3. Semen characteristics varied throughout the reproductive season reaching peak concentration in June-July. 4. The vacuum method proved to be the most efficient and was a low stress, restraint-free method for collecting ostrich semen.

It is well documented that prolactin (PRL) release and PRL gene expression in birds are controlled by the tonic stimulation of hypothalamic vasoactive intestinal peptide (VIP). However, there is good evidence that dopamine (DA) exerts both stimulatory (at the hypothalamic level) and inhibitory (at the pituitary level) effects on PRL secretion. The interactions between VIP and DA in the regulation of PRL gene transcription are not known. This study was designed to examine the effects of a D(2) DA receptor agonist (D(2)AG; R(-)-propylnorapomorphine HCl) on basal and VIP-stimulated PRL gene transcription rate, PRL mRNA steady-state levels, PRL mRNA stability and PRL release from cultured turkey anterior pituitary cells. The D(2)AG (10(-)(10) M) completely inhibited the stimulatory effect of VIP (10(-)(7) M) upon nascent PRL mRNA as determined utilizing a nuclear run-on transcription assay. To examine further the effect of the D(2)AG on PRL mRNA post-transcriptional events, anterior pituitary cells were treated with different concentrations of D(2)AG (10(-)(12)-10(-)(4) M). Semi-quantitative RT-PCR and RIA were performed to determine the levels of PRL mRNA and PRL content in the medium respectively. The results show that D(2)AG inhibited VIP-stimulated PRL mRNA steady-state levels as well as basal and VIP-stimulated PRL release, effects which were diminished by the D(2) DA receptor antagonist, S(-)-eticlopride HCl (10(-)(10) M). Actinomycin D (5 microg/ml), an inhibitor of mRNA synthesis, was used to assess the effect of D(2)AG on PRL mRNA stability in response to VIP. The stimulatory effect of VIP on PRL mRNA stability was completely negated by the D(2)AG (from a half-life of 53.0+/-2.3 h in VIP-treated cells to 25.5+/-1.6 h in D(2)AG+VIP-treated cells, P

Artificial illumination, including light quality, is important in modern meat-type poultry management. In the present study, the effect of in ovo monochromatic green light photostimulation on posthatch growth of turkey poults was investigated. In experiment 1, 182 turkey eggs were divided into two light treatment groups (n = 91). The first group was intermittently photostimulated (3 min on and 3 min off) with green light provided by five light-emitting diodes (LED) per egg at 0.1 W/m2 at the upper eggshell surface. The second group was incubated in the dark and served as the control. Posthatch BW were recorded at 0, 2, 6, 13, 20, 28, 35, and 59 d of age. A heavier BW, occurring at 28 d of age and persisting until the end of the experiment (59 d of age), was observed in the in ovo green light stimulated females as compared to their corresponding controls. In experiment 2, 273 turkey eggs were divided into three light treatment groups (n = 91). The first group was intermittently photostimulated (15 min on and 15 min off) with green light provided by seven LED per egg at 0.14 W/m2. The second group was photostimulated with white light provided by one mini-incandescent lamp per egg at light intensity and schedule similar to the first group. Eggs of the third group were incubated in the dark and served as controls. Posthatch BW were recorded at 0, 7, 14, 28, 42, 56, and 79 d of age. No differences were found among the BW of males incubated under different light conditions. As in experiment 1, female turkeys with stimulated green light in ovo had greater BW compared to their corresponding control and white light groups from 28 d of age until termination of the experiment at 79 d of age. Breast muscle weight was greater in female turkeys incubated under green light when compared to white and dark incubation treatment groups. We suggest that in ovo green light photostimulation enhances the posthatch BW of female turkey poults.

Ambient temperature modulates prolactin (PRL) secretion in birds. It is not known whether this modulation directly affects the PRL controlling mechanism(s) or whether it indirectly influences them through the onset of sexual maturity and/or the onset of incubation behavior. These experiments were designed to investigate the effect of elevated (32 degrees ) and reduced (10 degrees ) ambient temperatures on PRL secretion. Somatically mature, ovariectomized female turkeys were used to avoid the confounding effects of reproductive stage, nesting, and egg stimuli on PRL secretion. Hens were ovariectomized 5 weeks before, on the day of, or 10 days after the inception of photostimulation. Temperature treatments included chronic exposure (5 weeks) to 32 or 10 degrees and acute exposure (i.e., temperature was reversed from 32 to 10 degrees or from 10 to 32 degrees on or after the day of photostimulation). Chronic exposure to either 32 or 10 degrees had no effect on the rise in serum PRL that followed photostimulation in both sham-operated controls and ovariectomized hens. Acute exposure to 10 or 32 degrees altered the photoperiodically stimulated rise in plasma PRL. Birds switched from 10 to 32 degrees showed a significantly greater PRL increase than birds shifted from 32 to 10 degrees. Ovariectomy enhanced the PRL response to the gonadal stimulating photoperiod. The effect was most pronounced in hens photostimulated prior to ovariectomy. These findings suggest that ambient temperature and/or ovariectomy have a modulating effect on the PRL response to long days.

Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-R(intact)) showed 98% and 72-75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-R(insert)) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-R(insert) isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-R(trunc)) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.

Circulating prolactin (PRL) levels increase when dynorphin is infused into the turkey brain. This study tested the hypothesis that centrally infused dynorphin requires an intact vasoactive intestinal peptide (VIP) system in order to stimulate turkey PRL secretion. It also investigated the roles of the dopaminergic and serotonergic systems in dynorphin-induced PRL release. Drugs were infused into the third ventricle of anesthetized laying turkeys via stereotaxically guided cannulae and circulating blood was assayed for changes in PRL levels. When a highly selective ĸ opioid receptor antagonist was given prior to dynorphin injection, the PRL response to dynorphin was almost totally blocked. The coinfusion of either a serotonin (5-HT) or a D1 dopamine (DA) receptor antagonist with dynorphin prevented the increase in PRL observed in birds when dynorphin was infused alone. On the other hand, the ĸ opioid receptor antagonist failed to prevent the 5-HT-induced release of PRL. In hens actively immunized against VIP, infused dynorphin was unable to increase plasma PRL levels and infused VIP gave a muted PRL rise, while large increases in PRL were seen in nonimmunized birds receiving the same infusions. These data show that: (1) dynorphin stimulates PRL secretion by activating ĸ opioid receptors in the avian hypothalamus, and (2) dynorphin, 5-HT, DA, and VIP stimulate avian PRL secretion via a common pathway expressing ĸ opioid, serotonergic, dopaminergic, and VIPergic receptors at synapses arranged serially in that functional order, with the VIPergic system as the final mediator (releasing factor).

The regulation of prolactin secretion during the reproductive cycle of seasonal breeding birds appears to be largely under the stimulatory influence of hypothalamic vasoactive intestinal peptide (VIP). However, the factors influencing VIP secretion, and hence prolactin release, in birds remain largely unexplored. Recent evidence has demonstrated that dopamine and dopamine receptors may affect VIP and prolactin release in birds. The differential expression of dopamine receptors on hypothalamic VIP-releasing neurons may affect the degree of prolactinemia observed during the reproductive cycle of birds. In order to examine this hypothesis, we used reverse transcription-polymerase chain reaction to quantitate the levels of D1 and D2 dopamine receptor subtype mRNAs in the brain of the domestic turkey hen during the reproductive cycle. No significant difference in hypothalamic expression of D1 or D2 dopamine receptor subtypes during the reproductive cycle was observed. However, pronounced differences in D1D and D2 mRNAs were detected in cortex and cerebellum. Interestingly, there was a dramatic increase in pituitary D1D receptor mRNA during the reproductive stages of laying and incubation of eggs, which paralleled the hyperprolactinemic state of the turkey reproductive cycle. In addition, pituitary D2 receptor mRNA steadily increased throughout the reproductive cycle. In light of these observations, a modified hypothesis regarding the effects of dopamine on prolactin secretion is discussed.

The reverse transcriptase-polymerase chain reaction (RT-PCR), in combination with 5' and 3' rapid amplification of cDNA ends (RACE), was used to clone a G protein-coupled receptor from turkey brain mRNA. This cDNA clone has an open reading frame of 1,311 base pairs encoding a 436-residue protein with seven transmembrane-spanning domains and exhibits high homology with previously cloned mammalian D2 dopamine receptors. Northern blot analysis of turkey brain mRNA detected an approximate 2.4-kb transcript. RT-PCR and subsequent nucleotide sequence analysis of turkey brain and peripheral tissue mRNA also demonstrated the presence of an alternatively spliced mRNA corresponding to the predicted D2 short isoform. RT-PCR experiments demonstrated a widespread distribution of alternatively spliced D2 dopamine receptor transcripts throughout the turkey brain and in select peripheral tissues as well. In situ hybridization experiments detected strong autoradiographic signals over much of the turkey telencephalon, diencephalon, mesencephalon, cerebellum, pituitary, and pineal gland. Dopamine has several important functions as a neurotransmitter and hormone in mammals and may have similar actions in avian species. The cloning and tissue distribution of the D2 receptor subtype should enable the investigation of any functional role dopamine and dopamine receptors exert on the physiology and behavior of birds.