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Activation of temperature-dependent flagellar movement of demembranated fowl spermatozoa: involvement of an endogenous serine protease.

In the presence of ATP, the motility of demembranated fowl spermatozoa was vigorous at 30 degrees C, but negligible at 40 degrees C. Motility could be restored at 40 degrees C by the addition of 10-100 ng trypsin ml-1. Chymotrypsin also stimulated the motility, but neither papain nor carboxypeptidase B appreciably affected motility. Conversely, at 30 degrees C sperm motility was inhibited by aprotinin or phenylmethylsulfonyl fluoride. These results suggest that endogenous protease, presumably serine protease, activity is instrumental in the regulation of fowl sperm motility. It seems likely that the site of action of this protease is axonemal, but a direct effect of added protease on dynein ATPase activity could not be demonstrated.

Adenosine Triphosphatases↗

Regulation of flagellar motility of fowl spermatozoa: evidence for the involvement of intracellular free Ca2+ and calmodulin.

The possible role of intracellular free Ca2+ and calmodulin in the regulation of fowl sperm motility was investigated using an intracellular Ca2+ chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) and calmodulin antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and trifluoperazine. Intact fowl spermatozoa maintained vigorous movement in a Ca(2+)-free medium at 30 degrees C. In contrast, the motility of spermatozoa loaded with BAPTA/AM was negligible at 30 degrees C, but could be instantly restored by the addition of 2 mmol CaCl2 l-1. At this time, the intracellular free Ca2+ concentrations increased from 0 to about 100 nmol l-1, as measured by the fluorescent Ca2+ indicator fura-2. At 40 degrees C, neither control nor BAPTA/AM-treated spermatozoa were motile, but the motility of both kinds of spermatozoa was restored by the subsequent addition of 2 mmol CaCl2 l-1. Even in the presence of 2 mmol CaCl2 l-1, the addition of W-7 and trifluoperazine inhibited the motility of intact spermatozoa at 30 degrees C and 40 degrees C, and induced a concomitant decrease in the rate of oxygen consumption and ATP concentration, suggesting that energy depletion might be involved in the inhibition of motility. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of W-7 and trifluoperazine at 30 degrees C. The addition of W-5, a weaker antagonist, did not appreciably affect the motility of either intact or demembranated spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Possible role of protein kinase C in regulation of flagellar motility and intracellular free Ca2+ concentration of fowl spermatozoa.

The presence and possible role of protein kinase C in the regulation of fowl sperm functions were investigated. Immunoblot analysis of sperm extract using antibody to protein kinase C revealed a crossreacting protein of approximately 80 kDa. As the concentration of the protein kinase C activators N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) or 1-oleoyl-2-acetylglycerol (OAG) was increased, the motility of intact spermatozoa at 30 degrees C was reduced. However, this inhibition of motility was reversed by reducing the concentrations of activators. Even in the presence of 1 mmol CaCl2 l-1, the addition of SC-9 and OAG inhibited the motility of intact spermatozoa. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of SC-9 or OAG at 30 degrees C. However, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C inhibitor, did not appreciably affect the motility of either intact or demembranated spermatozoa at 30 degrees C. At 40 degrees C, both intact and demembranated spermatozoa were almost immotile in the presence or absence of the activators or inhibitor. Intracellular free Ca2+ concentrations, measured by means of a fluorescent Ca2+ indicator, fura-2, gradually increased after the addition of SC-9 and OAG, but no changes were observed in H-7-treated spermatozoa. These results suggest that endogenous protein kinase C is present in the cytoplasmic matrix or the membrane, but is not retained in the axoneme, and that the activation of this enzyme may contribute to a decrease in the flagellar movement of fowl spermatozoa.

Animals↗

Inhibition of temperature-dependent immobilization of fowl spermatozoa at body temperature by an increased intracellular pH.

Fowl sperm motility was measured by altering the extracellular pH (pHe) at 30 degrees C and 40 degrees C. At 30 degrees C, the motility of intact spermatozoa was vigorous in a medium in the wide pHe range of 7.3-10.1. In contrast, intact spermatozoa were almost immotile at 40 degrees C in medium below a pHe of 8.1. However, the motility could be restored by increasing the pHe; maximum motility was obtained in medium at pHe 9.4. Stimulation of the motility of demembranated spermatozoa at 40 degrees C was also observed with an increased pHe. However, demembranated spermatozoa at 40 degrees C that had been stimulated by increasing the pHe lost their motility when 1 mmol CaCl2 l-1 was added. Motility was restored by the subsequent addition of 2 mmol EGTA l-1. At a high pHe at 40 degrees C, the flagellar ATPase activity of crude dynein extract was not affected, regardless of the addition of CaCl2 or EGTA. The intracellular pH (pHi) of intact spermatozoa, estimated by measuring the accumulation of 9-aminoacridine fluorescence, increased with increasing pHe at both 30 degrees C and 40 degrees C. These results demonstrate that the reversible temperature-dependent immobilization of fowl spermatozoa at 40 degrees C is inhibited by an increased pHi. Furthermore, it is possible that the effects of the increased pHi may not act directly on dynein ATPase activity, but are mediated by a Ca(2+)-related substance(s) on the axoneme.

Animals↗

Regulatory mechanisms of fowl sperm motility: possible role of endogenous myosin light chain kinase-like protein.

The motility of both intact and demembranated fowl spermatozoa was vigorous at 30 degrees C, but decreased markedly following the addition of 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), a specific inhibitor of myosin light chain kinase (MLCK). Furthermore, the presence of a MLCK substrate peptide also inhibited the motility of demembranated spermatozoa at 30 degrees C. In contrast, the addition of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8) or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride (HA1004), specific inhibitors of cAMP-dependent protein kinase, did not appreciably affect the motility of either intact or demembranated spermatozoa. Cyclic AMP-dependent protein kinase substrate peptides were also ineffective for the inhibition of motility of demembranated spermatozoa at 30 degrees C. Immunoblotting of sperm extract, using an antibody to MLCK, revealed two major crossreacting proteins of 130 kDa and 61-64 kDa, which corresponded to the molecular mass of MLCK. In addition, immunogold particles, which reacted with the anti-MLCK antibody, were observed around or on the axoneme at the ultrastructural level. These results suggest that the phosphorylation of axonemal protein(s) by MLCK, or a MLCK-like protein, rather than by cAMP-dependent protein kinase, may be involved in the maintenance of fowl sperm motility at 30 degrees C.

Animals↗

Differential sensitivity of ovarian follicles to gonadotrophin stimulation in broiler and layer lines of domestic fowl.

Differences in the reproductive potential of female broiler parent and layer fowl are related to differences in the recruitment of follicles to the preovulatory hierarchy. Broiler and laying hens were fed ad libitum or a restricted diet during rearing and ad libitum after sexual maturity. The sensitivity of thecal tissue, from ovarian follicles of different sizes, from layer and broiler fowl to stimulation by pregnant mares' serum gonadotrophin (PMSG) was determined to test the hypothesis that differences in egg production are associated with changes in the sensitivity of ovarian follicles to stimulation by gonadotrophin. The activity of ornithine decarboxylase 0.5-4 h after injection of 200 iu PMSG kg-1 body mass was assessed in thecal tissue from small white follicles 2-3 and 5-6 mm in diameter and in the smallest preovulatory yellow follicle. Basal activity was similar in layers and broiler breeders. PMSG-stimulated ornithine decarboxylase activity in follicles of 5-6 mm diameter was greater in broiler breeders than in layers. There was no difference between the lines in PMSG-stimulated ornithine decarboxylase activity in follicles of 2-3 mm diameter or in small yellow follicles. It was concluded that the low egg production and high prevalence of eggs with defective shells in broiler parents compared with layers may be related to the increased sensitivity of follicles to endogenous gonadotrophins at the critical stage of recruitment to the preovulatory hierarchy.

Animal Nutritional Physiological Phenomena↗

Stimulation of motility and respiration of intact fowl spermatozoa by calyculin A, a specific inhibitor of protein phosphatase-1 and -2A, via a Ca2+-dependent mechanism.

Intact fowl spermatozoa became almost immotile at 40 degrees C. In contrast, the presence of 10-1000 nmol calyculin A l-1, a specific inhibitor of protein phosphatase-1 (PP1) and -2A (PP2A), permitted activation of sperm motility in a dose-dependent manner. Calyculin A also stimulated the rate of oxygen consumption by spermatozoa, and induced a concomitant decrease in ATP concentrations, suggesting a coupling of ATP hydrolysis to the rate of oxidative phosphorylation. However, the motility and oxygen consumption of spermatozoa loaded with an intracellular Ca2+ chelator, 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM), were not stimulated by calyculin A alone, but only after the subsequent addition of 2 mmol CaCl2 l-1. These results suggest that inhibition of the activities of endogenous PP1 and PP2A may stimulate the motility and metabolic activity of fowl spermatozoa at 40 degrees C via a mechanism that requires intracellular free Ca2+.

Animals↗

Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro.

At the avian body temperature of 40 degrees C, intact fowl spermatozoa require Ca(2+) for the initiation of motility and a combination of both Ca(2+) and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1-100 micromol/l, neither PD 150606 (a Ca(2+)-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca(2+)-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca(2+) and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca(2+), as well as motility initiated by calyculin A -- a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 degrees C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

Acrosome Reaction↗

Influence of heat and vibration on the movement of the northern fowl mite (Acari: Macronyssidae).

Heat and vibration are common host-generated cues that ectoparasites use to orient to hosts. Three experiments evaluated effects of heat and vibration on the movement of northern fowl mite, Ornithonyssus sylviarum (Canestrini & Fanzago). Individual arrested mites in an isolation chamber always initiated movement (walking) after substrate vibration (7.8-min walking duration), but only initiated movement 50% of the time (2.8-min walking duration) upon exposure to a 3 degrees C heat fluctuation. Heat fluctuation in combination with vibration extended the period of activity by approximately 50% (11.6-min walking duration) compared with activity initiated by vibration alone. Mites with longer time off-host moved for shorter durations. In a choice test, individual mites consistently moved closer to a 35 degrees C heat source 1 or 6 mm away, but not to a heat source 11 mm away. In a circular arena, mites were able to orient accurately to a 35 degrees C heat source and reached the arena edge almost 4 times faster (11.2 s) than mites without a heat source (41.2 s). These results suggest that northern fowl mite is capable of directed thermo-orientation, as well as modulation of activity depending on the type of sensory information perceived. The adaptive significance of this orientation for a "permanent" ectoparasite is discussed.

Acaridae↗

Protection conferred by a live Salmonella Enteritidis vaccine against fowl typhoid in laying hens.

Fowl typhoid is under control in poultry farms of developed countries, but it still endemically subsists in commercial laying hen farms of some countries. It has been demonstrated that Salmonella live vaccines can elicit cross-immunity against members of the same Kauffmann-White scheme serogroup. In this work, we explored the protection conferred by TAD Salmonella vac E, a live Salmonella enterica serovar Enteritidis vaccine, against fowl typhoid. Three groups of laying hens were vaccinated with different vaccination schedules starting on the first day of life, and afterwards were infected with 2 x 10(5) CFU of a virulent Salmonella Gallinarum strain, either at wk 28 or wk 52. Mortality, fecal shedding, and organ invasion of Salmonella Gallinarum were assessed. In this work we demonstrated that this Salmonella Enteritidis vaccine is able to cross-immunize against Salmonella Gallinarum. At wk 28, hens vaccinated with three oral doses or with two oral doses combined with one subcutaneous dose were protected by the vaccine. At wk 52, when hens were infected 36 wk after the final immunization, the vaccine was not able to confer protection. Thus, revaccination every 3 mo would be highly recommended. In countries where Salmonella Gallinarum subsists together with Salmonella Enteritidis, control programs should include vaccination of laying hens using safe attenuated Salmonella strains.

Animals↗

Effect of luteinizing hormone on progesterone secretion in vitro by the granulosa cells of the domestic fowl (Gallus domesticus).

During short periods of incubation (3 h) the secretion of progesterone by granulosa cells from the largest preovulatory follicle of the fowl was higher (160 pmol/micrograms DNA) with ovine LH in the medium than without it (60 pmol/micrograms DNA). Granulosa cells from follicles collected 24 and 48 h before their expected ovulation secreted progesterone at similar rates to cells from the largest follicle which was likely to ovulate within 5 h. The identity of progesterone was confirmed by physicochemical methods. After granulosa cells had been incubated with LH in Medium 199 for 24 h, the concentration of progesterone in the medium was 1.65 mumol/l whereas oestrone and oestradiol were present at concentrations of 254 and 199 pmol/l respectively. The results indicate that the larger yellow yolk-filled follicles of the ovarian hierarchy in the domestic fowl contribute to the preovulatory surge of progesterone which has been observed in the peripheral blood.

Animals↗

Effect of gonadectomy on the concentrations of catecholamines in discrete areas of the diencephalon of the domestic fowl.

Concentrations of dopamine, noradrenaline and adrenaline in discrete areas of the diencephalon in male and female domestic fowl were correlated with changes in the plasma concentrations of LH induced by gonadectomy. Gonadectomized birds of both sexes exhibited raised plasma concentrations of LH and in castrated cockerels the daily administration of testosterone propionate was completely effective in preventing the postcastration rise in LH. Although no significant alterations in the brain concentrations of noradrenaline or adrenaline were observed in cockerels, the concentration of dopamine in the paraventricular nucleus (PVM), dorsomedial thalamic nucleus and mediobasal hypothalamus (MBH) were significantly raised in castrated compared with sham-operated birds by 136, 182 and 52% respectively. In each case the increase was partially suppressed by testosterone replacement therapy. In pullets, ovariectomy resulted in significant increases in the concentrations of dopamine (83%) and noradrenaline (78%) in the MBH and noradrenaline (35%) and adrenaline (34%) in the PVM. These findings suggest that in the fowl at least part of the negative feedback effect of gonadal steroids on LH secretion may be mediated by catecholaminergic mechanisms at the level of the hypothalamus.

Animals↗

Diminution of thyrotrophin releasing hormone-induced growth hormone secretion in adult domestic fowl (Gallus domesticus).

Age-related changes in the response of GH to administration of thyrotrophin releasing hormone (TRH) have been investigated in the domestic fowl. In two strains of chicken the i.v. administration of TRH (10 microgram/kg) to 4-week-old male and female birds markedly increased (greater than 100 ng/ml) the plasma GH concentration within 10 min of treatment and the concentration remained higher than the pretreatment level for at least a further 20 min. Saline (0.9%) administration had no effect on GH secretion in comparable groups of control birds. The same dose of TRH had no effect on plasma GH concentrations in adult (greater than 24-week-old) laying hens or cockerels. The administration of TRH at doses of 0.1-100 microgram/kg (i.v.) or 0.39-50 microgram/bird (s.c.) also had very little, if any, effect on GH secretion in laying hens. In laying hens slight increases (10-20 ng/ml, P less than 0.05) inthe plasma concentrations of GH were observed in one experiment 60 min after the s.c. injection of 100 microgram TRH, and in another 60, 90 and 120 min after the serial s.c. injection of TRH (100 microgram/bird) every 30 min over a 150 min period. The poor GH response to TRH of the adults to TRH stimulation was not due to high circulating concentrations of endogenous gonadal steroids, as surgical gonadectomy had no effect on the GH response to TRH. These results suggest maturational differences in the control of GH secretion in the fowl.

Age Factors↗

Role of serotonin in the regulation of growth hormone and prolactin secretion in the domestic fowl.

Plasma levels of GH and prolactin were measured by radioimmunoassay in male domestic fowl treated with centrally active agents. p-Chlorophenylalanine (pCPA) did not have an effect on tonic levels of prolactin but led to a significant rise in circulating GH concentrations. The three serotonin receptor antagonists tested, methysergide, SQ-10631 and cyproheptadine, each resulted in a significant reduction in plasma prolactin while markedly increasing plasma GH levels. Administration of 5-hydroxytryptophan led to a rise in plasma prolactin and a drop in plasma GH levels in untreated birds or in animals pretreated with pCPA. The serotonin receptor agonist, quipazine, resulted in a marked increase in plasma prolactin and a marked reduction in plasma GH concentrations in untreated birds. In pCPA-pretreated animals quipazine was no longer effective in altering plasma prolactin levels but still caused a significant drop in circulating levels of GH. These results suggest that in the young male domestic fowl serotonin has a stimulatory role in the regulation of prolactin and an inhibitory role in the regulation of GH secretion.

5-Hydroxytryptophan↗

Abnormalities in the plasma concentrations of thyroxine, tri-iodothyronine and growth hormone in sex-linked dwarf and autosomal dwarf White Leghorn domestic fowl (Gallus domesticus).

Plasma concentrations of GH were high in young birds (2-12 weeks old) and low in older birds (15 and 18 weeks old) in the control Cornell K strain of White Leghorn fowl. There was little variation in the plasma concentrations of tri-iodothyronine (T3) and thyroxine (T4) with age in this strain. Similar patterns of plasma hormone concentrations with age were observed in autosomal recessive dwarf chickens. However, the plasma concentrations of T3 and T4 tended to be depressed while that of GH was raised. There was little age-related change in the plasma concentrations of GH, T3 and T4 in sex-linked recessive dwarf chickens. Throughout growth and maturation the plasma concentrations of T3 in the sex-linked dwarf birds were less 40% of those observed in the control strain. Plasma concentrations of T4 tended to be raised relative to the control in the sex-linked dwarf at each age examined. The plasma concentration of GH was raised only in older (12, 15 and 18 weeks old) sex-linked dwarfs. Liver T4-5' monodeiodinase activity was slightly depressed in autosomal dwarf chickens and very low in the sex-linked dwarf fowl. Neither autosomal nor sex-linked dwarfism appears to be due to hypopituitarism. It is probable that the depressed liver 5' monodeiodinase activity and the concomitant low plasma concentrations of T3 are a causative factor in the reduced growth and stature of the sex-linked dwarf chicken.

Animals↗

Dopaminergic inhibition of prolactin release from pituitary glands of the domestic fowl incubated in vitro.

Anterior pituitary glands from broiler fowl were incubated by themselves, with hypothalamic tissue or with thyrotrophin releasing hormone (TRH) in medium containing dopamine and its antagonist pimozide. The presence of hypothalamic tissue or TRH resulted in a stimulation of release of prolactin. Neither dopamine nor pimozide affected prolactin release directly from the pituitary gland. Dopamine inhibited the release of prolactin stimulated by hypothalamic tissue or TRH, in a concentration-dependent fashion. Pimozide diminished the response to dopamine. After pituitary glands were preincubated for 20 h in medium containing oestradiol-17 beta, the basal release of prolactin was enhanced as was the response to TRH. Both basal and TRH-stimulated release of prolactin from the oestrogen-primed pituitary glands was inhibited by dopamine, an effect blocked by pimozide. Hypothalami from broiler fowl were incubated for up to 8 h in medium containing dopaminergic drugs and pituitary glands were incubated in this medium, alone or with pimozide. As indicated by the prolactin released by the pituitary glands, the hypothalami appeared to secrete prolactin-releasing activity in a time-related fashion. Dopaminergic activity was also present in the hypothalami, since pimozide enhanced the prolactin-releasing activity of the medium. Dopamine apparently inhibited and pimozide stimulated the secretion of releasing activity from the hypothalamus. These results suggest that dopamine inhibits release of prolactin directly from the pituitary gland only when prolactin secretion is high. The hypothalamus secretes at least two factors regulating prolactin secretion, a prolactin-releasing factor and a dopaminergic prolactin-inhibiting factor. Dopamine may also play an inhibitory role in the regulation of secretion of the prolactin-releasing factor.

Animals↗

Changes in aromatase activity in small ovarian follicles of the domestic fowl (Gallus domesticus) during growth and atresia.

The tritium release assay for measuring aromatase activity was adapted to measure oestrogen production in intact small ovarian follicles of the domestic fowl. The activity was measured in three types of follicle classified as normal, atretic or grossly atretic. Normal follicles were distinguished from atretic by the presence of small haemorrhages on the surface of the atretic follicles. Grossly atretic follicles were identified by their deformed shape. The aromatase activity in normal and atretic follicles was related to follicular weight, the activity in atretic follicles being less than that in normal follicles. The aromatase activity in grossly atretic follicles was independent of follicular weight. The enzyme activity in this group of follicles was significantly less than that in either normal or atretic follicles. The significance of these results is discussed in relation to the induction of atresia within the small ovarian follicles of the domestic fowl.

Animals↗

Somatostatin immunoneutralization stimulates thyroid function in fowl.

The influence of somatostatin on thyroid function has been examined in immature domestic fowl passively immunized with somatostatin antiserum. Plasma thyroxine (T4) and tri-iodothyronine (T3) concentrations were markedly increased within 10 min of antisomatostatin administration and remained raised for at least 5 h. The increases in the T3 and T4 concentrations following somatostatin immunoneutralization were directly related to the volume of antisera administered. The increase in the T3 concentration exceeded the increase in the T4 concentration, resulting in a T3 : T4 ratio greater than unity. While the raised T4 concentration began to decline 30 min after antisomatostatin administration, raised T3 concentrations were sustained for at least 2 h, and further increased the plasma T3 : T4 ratio. These results demonstrate that somatostatin immunoneutralization stimulates thyroid function in fowl. The magnitude and rapidity of the thyroidal responses to somatostatin immunoneutralization suggests that they occur independently of the hypothalamic-pituitary-thyroid axis. Somatostatin appears to exert a tonic inhibitory control on avian thyroid function, possibly by effects mediated at the thyroid gland to inhibit T4 release and by peripheral effects to suppress the conversion of T4 and T3.

Animals↗