Defining characteristics of immersion carbon dioxide gas for successful euthanasia of neonatal and young broilers.

This study investigated how the carbon dioxide (CO2) concentration within a chamber affects the efficacy of CO2 euthanasia and how the efficacy of CO2 induction methods changes as birds age. In experiment 1, pairs of broiler chicks (n = 192; 0, 3, and 6 D of age) were immersed into a chamber prefilled with 70, 80, 90, or 100% CO2. For experiment 2, 3- and 6-day-old broiler chicks (n = 88) were immersed in pairs into 100% CO2 or exposed to CO2 gradual fill in a chamber with a displacement rate of 28% chamber volume per minute. Latency to performance of headshaking (HS) and gasping (GS) as potential indicators of distress, loss of posture indicative of insensibility, and the cessation of rhythmic breathing (CRB) and cessation of movement (COM) as the indicators of death were monitored (live focal sampling/video recordings). The duration and frequency of HS and GS were assessed. For both experiments, behavior data were analyzed for CO2 method and age (4 × 3 factorial). Age and CO2 concentration interacted for latency to CRB and COM, with longer latencies for 0-day-old chicks immersed into 70% CO2 than other concentrations and ages. CO2 concentration did not affect latency to HS, GS, or loss of posture but affected CRB and COM, with latencies longest for 70% and shortest for 90 and 100% CO2. Newly hatched chicks had a longer latency to CRB and COM and longer duration and frequency of distress behaviors than older chicks. At all ages, initiation of all behaviors occurred later with gradual fill compared to immersion. There was an increased duration and frequency of distress behaviors with gradual induction compared with immersion. Overall, immersion into 90 to 100% CO2 resulted in the shortest time to insensibility and death, with a decreased duration and frequency of distress behaviors. Chicks immersed into 70% CO2 had the longest duration of GS and time to death. Age affects the efficacy of CO2 euthanasia, with increasing age decreasing time to death and the duration and frequency of distress behaviors.

Evaluation of carbon dioxide induction methods for the euthanasia of day-old cull broiler chicks.

This study was conducted to evaluate the efficacy of 5 different CO2 euthanasia induction techniques for day-old cull chicks in minimizing distress and inducing a rapid loss of sensibility and death. Each induction treatment was characterized for concentration change over time, maximum concentration, and time to reach maximum. Sixteen chicks were euthanized with the gradual treatments to establish validity of treatment. Then, all 5 treatments were evaluated for effect on distress, insensibility, and death. Day-of-hatch cull chicks (n = 110) were euthanized in pairs by either immersion into 100% CO2 or gradual induction to 100% CO2 at displacement rates of 7, 14, 21, or 28% of chamber volume added per min (% vol/min). CO2 concentration was measured at chick level. Live focal observations and video recordings were used to assess latency to behavioral responses: head shaking (HS) and gasping (GS) as indicators of distress; loss of posture (LOP) as an indicator of insensibility; and cessation of rhythmic breathing (CRB) and movement (COM), indicating death. All behaviors occurred at the earliest with immersion compared to gradual treatments, and time between first signs of distress and LOP was shorter for immersion than gradual treatments. Gradual treatments showed a linear decrease in latency to HS, GS, and LOP as displacement rate increased. Latency to CRB decreased quadratically with increasing displacement rate, while COM decreased linearly. Within gradual treatments, HS and GS occurred at CO2 concentrations between 0.43 and 1.14%, LOP between 11.1 and 17.5%, while CRB and COM occurred between 61.8 and 78.4%. Overall, immersion induced distress, insensibility, and death significantly faster and with the shortest interval between distress and insensibility. For gradual treatment, insensibility and death occurred faster with increasing displacement rates. Behavioral signs of distress were observed with all treatments, and occurred at concentrations lower than those causing insensibility. In conclusion, immersion into 100% CO2 environment resulted in the shortest time of distress and fastest time to death compared to gradual displacement rates of any speed measured.

Melanin-concentration hormone updated functional considerations.

The melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced in hypothalamic perikarya whose fibers project to most regions of the brain and into the spinal cord. Its role as a neurohypophyseal color-change hormone is peculiar to teleost fish, but recent studies in mammals suggests that MCH itself, and other peptides derived from the same precursor, may participate in multiple functions in the central nervous system, modulating behavior and the perception of sensory information. Recent hybridization studies in mammals have greatly increased our understanding of the response of the MCH system to environmental factors, such as osomotic challenge, lactation, stress, and changes in corticosteroid levels. Further studies in lower vertebrates are needed to highlight the physiologically important functions that have led to the structural conservation of the MCH peptide during vertebrate evolution.

Melanin-concentrating hormone binding to mouse melanoma cells in vitro.

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 was replaced by Phe and Val19 by Tyr. The resulting monoiodinated [125I] [Phe13,Tyr19]-MCH radioligand was biologically active and led to the discovery of high-affinity binding sites on mouse B16-F1, G4F and G4F-7 melanoma cells. Saturation binding analysis with G4F-7 cells revealed 1090 MCH receptors per cell and a KD of 1.18 x 10(-10) mol/l. Receptors for MCH were also found on rat PC12 phaeochromocytoma cells, human RE melanoma cells and COS-7 cells. Competition binding analyses with other peptides such as alpha-MSH, NPY and PACAP demonstrated that MCH receptor binding is specific. rANF(1-28) was found to be a weak competitor of MCH, indicating topological similarities between MCH and rANF(1-28) when interacting with MCH receptors.

An immunological study of the secretory activity of neurons producing melanin-concentrating hormone in a teleost.

The melanin-concentrating hormone is a general vertebrate neurosecretory peptide which, in bony fish, serves as a neurohypophysial hormone influencing pigmentary changes in response to background colour. Young carp were reared for six months in white- or black-coloured tanks to determine how this would influence the development of the neurons producing the peptide. Cytological criteria and radioimmunoassay of tissue extracts showed that the background markedly influenced the synthetic activity of these neurons. In carp reared in black tanks, the perikarya were small and poorly granulated, with small nuclei and often undetectable nucleoli. Transfer of such fish to a white tank for six days caused no significant change in hormone content but cytological criteria suggested an increased activity of some of the neurons. In fish reared on a white background, over 50% of these neurons showed a greatly enhanced synthetic activity, while radioimmunoassays showed significantly higher concentrations of immunoreactive peptide in their hypothalami but not in their pituitary glands. After such fish were moved to black tanks for six days, the neuropeptide content of the hypothalamus and pituitary gland was significantly increased. Histologically, this was reflected in the amount of immunostainable granulation in both sites but cell nuclear size was not decreased. These changes are interpreted in terms of changes of hormone synthesis and release. The observations provide evidence that the activity of many but not necessarily all of the neurons producing melanin-concentrating hormone in the carp hypothalamus is controlled by background colour.

Ability of various factors to oppose the stimulatory effect of dibutyrylcyclic AMP on the release of melanocyte-stimulating hormone by the rat pituitary in vitro.

Various agents were tested for their ability to oppose the stimulatory effect of dibutyryl cyclic AMP on the release of the melanocyte-stimulating hormone from the rat neuro intermediate lobe in vitro. Only dopamine exhibited an inhibitory effect; serotonin, gamma-amino-butyric acid, tocinoic acid, tocinamide, the tripeptide Pro-Leu-Gly-NH2 and dibutyryl cyclic GMP were all ineffective.

The influence of repeated stress on the release of melanin-concentrating hormone in the rainbow trout.

Melanin-concentrating hormone (MCH) is a neurohypophysial peptide that induces pigmentary pallor in teleosts and which is released when the fish are placed on a white background. An additional effect of the peptide is the depression of ACTH and hence cortisol secretion during moderate stress. The present work on rainbow trout shows that plasma MCH concentrations, while unaffected by a single stress, are raised by repeated stress (1 ml saline injected i.p. without anaesthesia) and remain high for several hours thereafter. The response to stress is observed only in white-adapted fish and not in fish kept in black-coloured tanks, when MCH release is normally low. Plasma concentrations of MCH vary diurnally but stress induces an equivalent incremental rise in plasma MCH, whether administered in the middle or towards the end of the photophase. The stress-induced rise in MCH concentrations is prevented by treatment with dexamethasone. The results support the suggestion that the modulatory effect of MCH on the hypothalamopituitary-interrenal axis of fish might be enhanced under conditions of stress.

Seasonal and photoperiod-induced changes in the secretion of alpha-melanocyte-stimulating hormone in Soay sheep: temporal relationships with changes in beta-endorphin, prolactin, follicle-stimulating hormone, activity of the gonads and growth of wool and horns.

Blood plasma concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH), beta-endorphin (beta-END), prolactin and follicle-stimulating hormone (FSH), and associated changes in the size of the testes, and growth of the horns and pelage were measured in male (n = 8), castrated male (n = 5) and female (n = 9) Soay sheep. The animals were born in April and kept outdoors near Edinburgh (56 degrees N) during the first two years of life. In all groups there was a close association between the weekly changes in the plasma concentrations of alpha-MSH and beta-END; the molar ratio in mean concentrations was close to 1:1. The blood plasma concentrations of both hormones varied markedly with season with a 3- to 10-fold increase in concentrations from the minimum in winter to the maximum in autumn. The seasonal peak occurred in September in the first year of life as juveniles, and between July (males) and September (females) in the second year when the animals were sexually mature. The plasma concentrations of ACTH did not vary in parallel with the seasonal changes in the concentrations of alpha-MSH (measured only in males); the molar ratio for the concentrations of alpha-MSH:ACTH was 1:0.12. The seasonal increase in the concentrations of alpha-MSH occurred 1-3 months after the seasonal increase in the concentrations of prolactin and the associated growth in horns and pelage, and slightly before, or coincident with the seasonal increase in the concentrations of FSH and the growth in the testes. In a second experiment, the same parameters were measured in a group of adult male Soay sheep (n = 8) housed indoors under an artificial lighting regimen of alternating 16-week periods of long (16 h light: 8 h darkness) and short days (8 h light: 16 h darkness). In this situation, there was a clearly defined photoperiod-induced cycle in the plasma concentrations of alpha-MSH with a 25-fold increase from a minimum under long days to a maximum under short days. The concentrations of beta-END varied in close parallel with the changes in alpha-MSH, and the temporal associations with the changes in the other pituitary hormones were similar to those observed in animals housed outdoors. Overall, the results support the view that alpha-MSH is co-secreted with beta-END from the melanotrophs in the pars intermedia of the pituitary gland, and that the secretory activity of the melanotrophs changes markedly with season, increasing in summer and autumn, and decreasing in winter and spring.(ABSTRACT TRUNCATED AT 400 WORDS)

A black background facilitates the response to stress in teleosts.

This work examines the difference in responsiveness to stress which characterizes fish adapted to white and black backgrounds. Trout were maintained in black or white tanks for 2 weeks and then subjected to intermittent intense or moderate noise stress for periods between 1 h and 5 days, or to the stress of being injected daily with a large volume of liquid for 3 days. Plasma cortisol concentrations increased more readily and to a greater extent in fish from black tanks in response to moderate stress or brief intense stress. Dexamethasone suppressed the stress-induced rise of cortisol in white-adapted fish but was only partially effective in trout from black backgrounds. These differences in plasma cortisol between black- and white-adapted fish can be related to the different titres of plasma ACTH, apparently derived from the pars distalis. Removal of the neurointermediate lobe (NIL) from black-adapted eels markedly depressed the normal rise in plasma cortisol elicited by noise stress. It is suggested that products from the NIL may modulate the stress response of the hypothalamo-pituitary axis although other routes through which background colour could affect the pituitary responsiveness to stress are also considered. In several cases, stress also enhanced the secretion of MSH from the NIL.

Evidence for the participation of a melanin-concentrating hormone in physiological colour change in the eel.

The hormonal and nervous control of colour change in the eel has been investigated. The only bioactive forms of MSH found in eel pituitary extracts or secreted by eel pituitary cultures were forms of alpha-MSH; no beta-MSH was detected. After transfer of eels from a black to a white background, the melanin concentration in skin melanophores was accompanied by a rapid decline in plasma alpha-MSH titres. Hypophysectomy resulted in melanin concentration, and pituitary extracts injected into hypophysectomized eels caused melanin dispersion. This effect was eliminated if the pituitary extracts were first incubated with a specific alpha-MSH antiserum or if the antiserum was injected into the hypophysectomized eel. However, injection of alpha-MSH antiserum into intact, black-adapted eels failed to result in melanin concentration although the same antiserum was effective in causing pallor in black-adapted toads. Partially purified preparations of teleost melanin-concentrating hormone (MCH), free from catecholamines, induced melanin concentration when injected into black-adapted eels and this effect was significantly potentiated by injections of alpha-MSH antiserum. The denervation of melanophores on the pectoral fin had only a slight effect on the responses of the melanophores to humoral agents. It is concluded that the control of physiological colour change in the eel is largely hormonal, and involves the antagonistic effects of alpha-MSH and a melanin-concentrating agent which is probably MCH.

Differences in concentrations of plasma cortisol in the trout and the eel following adaptation to black or white backgrounds.

When rainbow trout (Salmo gairdneri) and eels (Anguilla anguilla) were kept in black tanks for 3-4 weeks, their plasma cortisol titres were about fourfold higher tha in fish kept in white tanks. In trout, the difference was apparent only under a long photoperiod of 16 h light: 8 h darkness, but in eels the difference was clear under both a long or short photoperiod (9.5 h light: 14.5 darkness). It is suggested that the increase in plasma cortisol seen in black-adapted fish is dependent on either ACTH or MSH secreted by the pars intermedia melanotrophs. No difference was seen in the total cortisol-binding capacity of the plasma nor in interrenal histology in trout from black or white backgrounds.

The effect of rearing rainbow trout on black or white backgrounds on their secretion of melanin-concentrating hormone and their sensitivity to stress.

Rainbow trout were reared in black or off-white coloured tanks for up to 18 months of age to achieve maximum differences in the synthesis of the neuropeptide, melanin-concentrating hormone (MCH). White-reared fish had greatly increased MCH concentrations in their pituitary glands, in their MCH perikarya and in the presumptive neuromodulatory fibres of the dorsal hypothalamus/thalamus when compared with black-reared and commercially reared trout. Following transfer to brighter white tanks, white-reared fish showed a significant increase in plasma MCH concentration and a reduction of MCH in the pituitary and MCH perikarya. The additional challenge of repeated stress further increased plasma MCH concentration in these fish and also reduced MCH in the dorsal hypothalamus/thalamus. In black-reared fish transferred to white tanks, plasma MCH concentrations were significantly raised after transfer, although they were lower after 11 days than in white-reared counterparts. Transfer from black to white background caused a fall in the MCH concentration in all regions--pituitary gland, perikarya and dorsal hypothalamus/thalamus; if transfer was accompanied by repeated stress, the hormone in the pituitary gland and MCH perikarya became so depleted that plasma MCH concentrations declined. Within each experimental situation (control, background transfer and transfer with stress) there was in inverse correlation between plasma MCH concentrations of black- and white-reared fish and the cortisol concentration. MCH had no direct effect on the secretion of cortisol by interrenal tissue but incubated hypothalami, in which endogenous MCH had been immunoabsorbed, provided evidence that MCH can depress the release of corticotrophin-releasing bioactivity.

Salmonid melanin-concentrating hormone inhibits corticotrophin release.

Melanin-concentrating hormone (MCH) is a neural peptide associated with colour change in fishes. We show here that it also inhibits corticotrophin (ACTH) secretion. Synthetic salmonid MCH at a concentration of 100pmol/l reduced the in-vitro release of ACTH by pars distales (pDs) taken from stressed trout. At lower concentrations (10pmol/l) the peptide inhibited CRF-41-induced secretion of ACTH by pDs removed from unstressed trout, while at higher concentrations (10nmol/l) it reduced the corticotrophic response of rat pituitary tissue to CRF-41.

Measurement of immunoreactive alpha-melanocyte-stimulating hormone in the blood of rainbow trout kept under various conditions.

Initial attempts to measure alpha-MSH in trout blood by radioimmunoassay proved unsuccessful due to apparent losses of immunoreactive hormone, especially in the plasma of trout adapted to a black background. An extraction protocol for alpha-MSH from plasma is described. Subsequent radioimmunoassay revealed progressive increases in immunoreactive alpha-MSH as trout became adapted to a black background. Plasma alpha-MSH titres were raised during fungal infection, and also showed significant differences in fish obtained from different fish farms.

The Biosynthesis of Melanin-Concentrating Hormone in Trout Kept Under Different Conditions of Background Colour and Stress, as Determined by an in vitro Method.

The aim of this study was to develop a method to monitor the synthetic activity of neurons which secrete the neurohypophysial melanin-concentrating hormone (MCH), a hormone implicated in two separate physiological roles in fish-pigmentary regulation and the response to stress. Trout hypothalamic fragments, containing the MCH neuronal cell bodies, were incubated in vitro in a medium including [(35) S]methionine. Labelled MCH-related products were separated by immunoprecipitation. Gel electrophoresis showed that radioactive methionine was incorporated into MCH precursors and into mature MCH, much as in vivo. Thus, de novo hormone synthesis continues in vitro. Trout reared at a fish-farm and adapted to black or white tanks for 39 days displayed nearly a 2-fold difference in their rate of methionine incorporation. Transferring fish from a black to a white background also doubled the rate of incorporation within 7 days and this rate increased only very slightly during the following 3 weeks. The rate of methionine incorporation by tissue from trout reared in black tanks was very depressed, and 4-fold lower than that of fish reared in white tanks, suggesting that very long-term adaptation to one or other background has increasingly marked effects on the activity and perhaps the number of synthetically active neurons. Stress also influenced the rate of methionine incorporation: a mild daily stress was stimulatory but more frequent stress had an inhibitory effect in many cases. The effects of daily dexamethasone administration were inconclusive. It is suggested that these differences in methionine incorporation reflect the relative rates of MCH synthesis in vivo, and that the method is useful to investigate conditions which modulate the biosynthesis of MCH in the trout.

The effect of photoperiod on plasma levels of melanin-concentrating hormone in the trout.

The neurohypophysial melanin-concentrating hormone, MCH, plays a role in adaptive colour change in teleost fishes inducing pallor when the fish is placed in pale-coloured surroundings. The present study shows that its plasma concentration, measured in groups of white-adapted fish, is not uniformly high throughout the day but follows a clear diurnal pattern. Over a 24 h cycle, plasma concentrations rise gradually during the morning to reach peak values around the middle of the photophase, after which they decline significantly before night. Lowest concentrations are observed during the dark period. This pattern was observed under a long photoperiod in summer and a short photoperiod in winter. The peak was shifted within a week of changing the onset of either light or dark. When dawn was delayed by 6 h for fish held under short photoperiod conditions, then peak concentrations were attained 6 h precociously. Fish from a long photoperiod placed in constant light showed a pattern of MCH release which approximated to the normal over the first 24 h period but plasma values then became raised and periodicity was no longer discernible. Plasma hormone concentrations were much reduced in trout kept in black coloured tanks in which nocturnal and daytime values differed, but significant differences during the photophase were not demonstrable. The results suggest that an illuminated white background can initiate the early morning release of MCH, and that an endogenous pacemaker underlies the pattern of MCH secretion.

The biosynthesis of melanin-concentrating hormone in a fish.

Abstract This work investigated the biosynthesis of a neurohypophysial hormone, melanin-concentrating hormone (MCH), in the trout. Sephadex G-75 chromatography showed the presence of several large MCH-immunoreactive molecules in hypothalamic and pituitary gland extracts, with different retention times on high-performance liquid chromatography from the mature MCH(1-17). About 10% of the total MCH-immunoreactivity in the hypothalamus was attributable to large molecular weight forms but these contributed less than 1% to the immunoreactivity in the neurointermediate lobe. Both [(35) S]methionine and [(3) H]leucine were injected into the hypothalamus near the MCH perikarya (nucleus lateralis tuberis region) of anaesthetized fish, after which the fish were killed at intervals of up to 8 h post-injection and the basal hypothalami, pituitary pars distales and neurointermediate lobes were extracted in acid. MCH-related immunoprecipitates from these extracts were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis or by Sephadex G-50 chromatography. The results show the incorporation of radiolabel into 15.3 K and 11.3 K precursors within 0.75 h, and their conversion, via several smaller intermediates, to a molecule resembling MCH(1-17). The results are discussed in relation to the known cDNA sequence of salmon MCH. Labelled MCH first appeared in the neurointermediate lobe 4 h after injection, giving an estimated transit rate of 0.4 mm/h.

The influence of gonadal steroids on pre-pro melanin-concentrating hormone mRNA in female rats.

Melanin-concentrating hormone (MCH) may have a regulatory role in the control of luteinizing hormone (LH) release. We have investigated if gonadal steroids induce changes in the expression of pre-pro MCH (ppMCH) that are associated with changes in the pattern of LH release. Using quantitative in-situ hybridization histochemistry we have determined the effect of administration of either oestradiol benzoate (5 microg/rat) or oestradiol benzoate followed 44 or 48 h later by progesterone (0.5 mg/rat) to ovariectomized rats on the expression of ppMCH in the medial and lateral zona incerta and the lateral hypothalamus. The prevalence of ppMCH transcripts in the intact female rat at 12.00 and 19.00 h on proestrus and the first day of dioestrus was also examined. Oestrogen reduced the intensity of hybridization signal for ppMCH mRNA and this was associated with both a decrease in the number of cells in which the message was detected in the medial zona incerta and a negative feedback effect on LH release in ovariectomized rats. Progesterone administration to oestradiol benzoate-primed rats did not alter the reduced expression in the medial zona incerta in spite of its positive feedback effect on LH release. We suggest that progesterone may act only on post-translational events. Expression in the MCH cell bodies of the lateral zona incerta were not affected but there was a transient decrease 4 h after progesterone treatment in the oestradiol benzoate-primed rats in expression in the lateral hypothalamus. No changes in ppMCH mRNA were seen in intact animals on proestrus or the first day of dioestrus indicating that gonadal steroids are not important in the modulation of ppMCH gene expression during the oestrous cycle. In other steroid-dependent physiological situations, however, oestrogen may influence the expression of ppMCH in a subpopulation of cell bodies in the medial zona incerta.

Influence of environmental colour and diurnal phase on MCH gene expression in the trout.

Melanin-concentrating hormone (MCH) gene expression in the brain of rainbow trout, reared and maintained in either pale or black-coloured tanks, was studied using in situ hybridization histochemistry. MCH transcripts were most prevalent in the magnocellular neurones of the nucleus lateralis tuberis (NLT), which project to the pituitary gland. They were also present, although at much lower levels, in dorsally projecting parvocellular neurones, sited more posteriorly above the lateral ventricular recess (LVR). In the NLT the most intense hybridization signal was seen over the pituitary stalk; above the LVR, the most active neurones were located caudally. In both the NLT and above the LVR, MCH hybridization signal was 4-fold stronger in white-reared fish than in black-reared fish. There was also a marked diurnal variation in MCH expression in both sites, with high levels at 16.00 h and lower levels at 04.00 h. The results show that gene activity in both hormonal (NLT) and neuromodulator/neurotransmitter (LVR) MCH neurones is induced by pale environmental colour and that MCH gene activity is subject to pronounced diurnal variation.

Diurnal changes in the expression of genes encoding for arginine vasotocin and pituitary pro-opiomelanocortin in the rainbow trout (Oncorhynchus mykiss): correlation with changes in plasma hormones.

Using quantitative in-situ hybridization, this study monitored diurnal changes in the abundance of the gene transcripts of two corticotropin-releasing peptides, arginine vasotocin (AVT) and isotocin in hypothalamic neurones, and of pro-opiomelanocortin (POMC) mRNA in the pituitary of the rainbow trout (Oncorhynchus mykiss). A significant diurnal pattern of gene expression was only displayed in the hypothalamus by the parvocellular AVT neurones of the preoptic nucleus. Abundance of AVT mRNA in these neurones was low at lights on (06.00 h), increased during the morning to reach a plateau of peak values between 14.00 h and 22.00 h, and then declined during the dark phase. This pattern was the inverse of that shown by plasma cortisol values. Changes in AVT transcript abundance are also considered in terms of the reported diurnal change in circulating AVT concentration. Pituitary and hypothalamic AVT peptide content did not change. Transcripts of both POMC genes (POMC-A and POMC-B) were monitored in pituitary corticotropes and melanotropes. Only POMC-A mRNA was detected in corticotropes where it showed no diurnal change in abundance. Transcripts of both POMC genes were found in the melanotropes, although, judging from autoradiographic intensity, POMC-A mRNA predominated. Both genes showed diurnal differences in their transcription with POMC-A mRNA showing peak values at 10.00 h and a nadir at 02.00 h, while POMC-B mRNA showed an inverse pattern. The results indicate that the two POMC genes can be independently regulated.