Targeted differential photostimulation alters reproductive activities of domestic birds.

Modern poultry production systems use environmentally controlled houses providing only artificial illumination. The role of light in reproduction of poultry depends on light quality (photoperiod, intensity/brightness, and spectrum), which enables us to provide custom-made illumination, targeted for the elevation of reproductive activities. Artificial targeted illumination significantly affects poultry reproduction. This phenomenon is based on the mechanism of light absorption in birds, which consists of two main components: the eye (retinal photoreceptors) and brain extraretinal photoreceptors. Several experiments on turkey hens and broiler breeder males and females have shown that photostimulation of brain extraretinal photoreceptors, while maintaining retinal photoreceptors under non-photostimulatory conditions, elevates reproductive activity by increasing egg production of hens and semen quality of roosters. In addition, we found acceleration in all gonadal axis parameters, leading to the acceleration in the production rate. Furthermore, we studied the role of retinal activation in gonadal axis suppuration and identified the role of serotonin in this phenomenon. As for today, several broiler breeder farms use targeted illumination based on our studies with excellent results.

The effect of monochromatic photostimulation on growth and development of broiler birds.

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.

Dopamine and mesotocin neurotransmission during the transition from incubation to brooding in the turkey.

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.

Melanopsin expression in dopamine-melatonin neurons of the premammillary nucleus of the hypothalamus and seasonal reproduction in birds.

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.

Photoperiodic modulation of clock gene expression in the avian premammillary nucleus.

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.

Serotonergic and catecholaminergic interactions with co-localised dopamine-melatonin neurones in the hypothalamus of the female turkey.

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.

Dopamine-melatonin neurons in the avian hypothalamus controlling seasonal reproduction.

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.

Rhythmic dependent light induction of gonadotrophin-releasing hormone-I expression and activation of dopaminergic neurones within the premammillary nucleus of the turkey hypothalamus.

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.

Rhythm-dependent light induction of the c-fos gene in the turkey hypothalamus.

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.

Identification of dopamine, gonadotrophin-releasing hormone-I, and vasoactive intestinal peptide neurones activated by electrical stimulation to the medial preoptic area of the turkey hypothalamus: a potential reproductive neuroendocrine circuit.

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.

Regulation of prolactin gene expression by vasoactive intestinal peptide and dopamine in the turkey: role of Ca signalling.

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.

The role of prolactin in reproductive failure associated with heat stress in the domestic turkey.

Reproductive failure associated with heat stress is a well-known 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 40 degrees C for 12 h during the photo-phase daily or maintained at 24-26 degrees C. 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.

Method for collecting semen from the ostrich (Struthio camelus) and some of its quantitative and qualitative characteristics.

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.

Effect of embryonic photostimulation on the posthatch growth of turkey poults.

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.

Molecular cloning and expression analysis of the turkey vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor whose activity in avian species is believed to be mediated by a specific VIP receptor (VIP-R). Circulating PRL levels are closely related to hypothalamic VIP immunoreactivity, hypothalamic VIP mRNA content, and hypophysial-portal blood VIP concentrations in turkeys. In the present study, a turkey VIP-R (tVIP-R) cDNA was cloned and its mRNA abundance was quantified in various tissues during different reproductive stages. The 2347-bp tVIP-R cDNA encoded a 457 amino acid protein, with a predicted Mr of 52 kDa. The full-length cDNA shares approximately 55% similarity with the mammalian VIP receptor-1. Northern blot analysis revealed that a major 2.7-kb transcript was expressed in laying hen pituitaries. Furthermore, two minor tVIP-R transcripts of 3.7 and 3.4 kb were observed. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using RNA from various turkey brain and peripheral tissues throughout the reproductive cycle. The steady-state levels of pituitary tVIP-R mRNA changed during the reproductive cycle, whereas mRNA expression in other tissues was not affected. The steady-state levels of tVIP-R mRNA were only affected in the pituitary, whereas mRNA expression in any of the other tissues examined following the immunization of turkeys against VIP were not affected.

Photostimulated prolactin release in the Turkey hen: effect of ovariectomy and environmental temperature.

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.

Molecular cloning and expression of turkey inhibin-alpha and -betaA subunits.

We isolated cDNA encoding turkey inhibin-alpha (tINH-alpha) and -betaA (tINH-betaA) subunits from the turkey ovary using reverse transcription-polymerase chain reaction (RT-PCR). The isolated alpha subunit and betaA subunit included the entire open reading frames encoding 329 and 424 amino acids, respectively. The amino acid sequences of mature tINH-alpha subunit and tINH-betaA subunit (12.6 and 12.9 kDa proteins, respectively), established via DNA sequence analysis, were highly conserved between the chicken and various mammals. Northern blot analysis revealed that the transcripts of tINH-alpha and tINH-betaA subunits were approximately 1.7 and 8.4 kb, respectively. In various stages of follicular development, tINH-alpha mRNA was highly expressed in small white follicles as compared to postovulatory and regressed follicles, whereas tINH-betaA mRNA was predominately expressed in preovulatory F5 follicles.

Active immunization with vasoactive intestinal peptide in turkey hens.

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.

Evidence for multiple prolactin receptor transcripts in the turkey.

Multiple prolactin receptor (PRL-R) mRNA transcript isoforms have been identified in mammals, but there are conflicting reports concerning the number of avian PRL-R isoforms. We hypothesized that multiple turkey PRL-R transcript isoforms exist and that PRL-R mRNA abundance may be related to reproductive status. Two turkey PRL-R cDNA fragments were generated using reverse transcriptase polymerase chain reaction (RT-PCR) that displayed a high degree of similarity to mammalian and avian PRL-R. Northern blot analysis of poly A+ mRNA hybridized to a turkey PRL-R riboprobe revealed a 3.1-kb band in the liver, oviduct, and testes. Additional 1.5- and 10.7-kb transcripts were found in the liver and testes, respectively. Hybridization of the same Northern blot to a chicken PRL-R probe verified the presence of a 3.1-kb transcript in all three tissues. A Northern blot was used to examine turkey PRL-R transcript isoform expression in laying hens. A 3.1-kb band was found in the pineal, infundibulum, magnum, isthmus, kidney, and intestine. In addition, 10.7- and 7.3-kb bands were detected in the pineal, magnum, isthmus, and intestine. Turkey PRL-R transcript isoforms were also examined throughout the reproductive cycle. The 10.7-, 7.3-, and 3.1-kb isoforms were detected in the oviduct, intestine, and pineal during each reproductive state. Turkey PRL-R mRNA levels were also compared during the reproductive cycle. Turkey PRL-R mRNA levels were greatest in laying hen pineal glands (P<0.05) and in incubating hen oviducts. This study provides the first evidence for multiple PRL-R mRNA transcript isoforms in turkeys.

Three different turkey luteinizing hormone receptor (tLH-R) isoforms I: characterization of alternatively spliced tLH-R isoforms and their regulated expression in diverse tissues.

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.