Effects of in ovo feeding of L-leucine on amino acids metabolism and heat-shock protein-70, and -90 mRNA expression in heat-exposed chicks.

Recently, we found that in ovo feeding of L-leucine (L-Leu) stimulated the metabolism of lipids and afforded thermotolerance in male Chunky broiler chicks. In this study, we investigated the effects of feeding L-Leu in ovo on the metabolism of amino acids and on the cellular stress response mainly in the central and peripheral tissues in neonatal male broiler chicks and partly in embryonic tissues. Chicks (9 d old) were exposed to high ambient temperature (HT: 35 ± 1°C) or control thermoneutral temperature (CT: 28 ± 1°C) for 180 min. The ambient temperatures were based on our recent reports and the recommendation of the Chunky broiler manual in which 28°C has been suggested as a normal ambient temperature for 5 to 9-d-old broiler chicks. In ovo feeding of L-Leu caused a significant (P < 0.05) decline in diencephalic arginine concentrations but it increased the diencephalic and plasma lysine concentrations when compared with the control chicks under HT. Notably, in ovo feeding of L-Leu significantly (P < 0.05) attenuated the increment of hepatic arginine compared with the control chicks under HT. Interestingly, in ovo feeding of L-Leu significantly (P < 0.05) attenuated the diencephalic gene expression of heat-shock protein (HSP) -70 and -90 in heat-exposed chicks. The gene expressions of mammalian target of rapamycin (mTOR) and its downstream genes (ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1)) in the central and peripheral tissues were not influenced in the chicks under heat stress. We found that the gene expressions of mTOR, S6K1, and 4E-BP1 were significantly (P < 0.05) stimulated only in the embryonic breast muscle, and not in the other embryonic tissues, by in ovo feeding of L-Leu. In conclusion, in ovo feeding of L-Leu caused a change in the metabolism of amino acids in response to heat stress in broiler chicks. Attenuated gene expressions of HSP-70 and -90 under heat stress further suggests that in ovo feeding of L-Leu may afford thermotolerance in broilers.

Oral administration of a medium containing both D-aspartate-producing live bacteria and D-aspartate reduces rectal temperature in chicks.

1. The aim of this study was to investigate the effects on the rectal temperature of young chicks of the oral administration of a medium that contained both live bacteria that produce D-aspartate (D-Asp) and D-Asp. 2. In Experiment 1, chicks were subjected to chronic oral administration of either the medium (containing live bacteria and 2.46 µmol D-Asp) or water from 7 to 14 d of age. Plasma-free amino acids as well as mitochondrial biogenic gene expression in the breast muscle were analysed. In Experiment 2, 7-d-old chicks were subjected to acute oral administration of the above medium or of an equimolar amount of D-Asp to examine their effect on changes in rectal temperature. In Experiment 3, after 1 week of chronic oral administration of the medium, 14-d-old chicks were exposed to either high ambient temperature (HT; 40 ± 1°C, 3 h) or control thermoneutral temperature (CT; 30 ± 1°C, 3 h) to monitor the changes in rectal temperature. 3. Chronic, but not acute, oral administration of the medium significantly reduced rectal temperature in chicks, and a chronic effect also appeared under HT conditions. 4. Chronic oral administration of the medium significantly reduced the mRNA abundance of the avian uncoupling protein (avUCP) in the breast muscle, but led to a significant increase in avian adenine nucleotide translocator (avANT) mRNA in the same muscle. 5. (a) These results indicate that the medium can reduce body temperature through the decline in avUCP mRNA expression in the breast muscle that may be involved in reduced mitochondrial proton leaks and heat production. (b) The increase in avANT further suggests a possible enhancement of adenosine triphosphate (ATP) synthesis.

Changes in free amino acid concentrations in the blood, brain and muscle of heat-exposed chicks.

An experiment was conducted to analyse the changes in free amino acid concentrations in the blood, brain and muscle of chicks in response to 15 or 30 min exposure to high ambient temperature (HT). Food intake and body weight were not affected, while rectal temperature was significantly increased by short-term HT exposure. Several free amino acid concentrations increased in the blood, brain and muscle even with short-term HT, whereas levels of a few amino acids declined significantly. As well as the nonessential amino acids, essential amino acids also significantly increased with exposure to HT. 3-Methylhistidine, a marker of proteolysis, significantly declined in the muscle of HT chicks, implying a reduction of protein breakdown under HT. These results indicate that alteration of protein metabolism may occur in chicks even with short-term heat exposure.

Lauroyl-L-aspartate decreased food intake and body temperature in neonatal chicks.

We hypothesized that the effects of L- and D-amino acids might be influenced when conjugated with fatty acid. Thus, the effects of oral administration of lauroyl-L-aspartate (Lau-L-Asp) as well as lauroyl-D-aspartate (Lau-D-Asp) were examined. In Experiment 1, oral administration of both Lau-L-Asp and Lau-D-Asp decreased food intake while L- or D-Asp did not influence food intake. Interestingly, only Lau-L-Asp decreased body temperature. Experiment 2 was conducted to determine whether non-conjugated mixture of L-Asp plus lauric acid has same effects under ad libitum feeding conditions. Lau-L-Asp decreased food intake and body temperature, but L-Asp plus lauric acid did not show any effect studied. In Experiment 3, we found that Lau-L-Asp declined food intake as well as time-dependently suppressed the body temperature in fasted chicks. However, L-Asp plus lauric acid did not show any effect. These results suggest that Lau-L-Asp may exert anorexigenic and hypothermic actions in chicks.

A new key neurohormone controlling reproduction, gonadotrophin-inhibitory hormone in birds: discovery, progress and prospects.

In vertebrates, the neuropeptide control of gonadotrophin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotrophin-releasing hormone (GnRH). Gonadal sex steroids and inhibin inhibit gonadotrophin secretion via feedback from the gonads, but a hypothalamic neuropeptide inhibiting gonadotrophin secretion was, until recently, unknown in vertebrates. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH acts on the pituitary and GnRH neurones in the hypothalamus via a novel G-protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotrophin release and synthesis. The pineal hormone melatonin is a key factor controlling GnIH neural function. GnIH occurs in the hypothalamus of several avian species and is considered to be a new key neurohormone inhibiting avian reproduction. Thus, the discovery of GnIH provides novel directions to investigate neuropeptide regulation of reproduction. This review summarises the discovery, progress and prospects of GnIH, a new key neurohormone controlling reproduction.

Changes in the responsiveness of hen anterior pituitary to cLHRH-II during induced molting.

The goal of this study was to determine whether the responsiveness of the anterior pituitary to cLHRH-II in relation to luteinizing hormone-beta subunit (LHbeta) mRNA expression is improved by induced molting. White Leghorn hens were subjected to induced molting by feed withdrawal for 4 days. The anterior pituitaries were collected from hens of pretreatment (PT), 1 day after resumption of feeding (1DRF) and on the day of resumption of laying (RL). They were processed for organ culture in the medium with or without cLHRH-II, followed by reverse transcription and competitive PCR. When pituitary tissues were incubated without cLHRH-II, the expression of LHbeta mRNA did not show any significant difference between PT and RL group hens. In contrast, the expression of LHbeta mRNA in the pituitaries that were incubated with cLHRH-II was significantly greater in RL group hens than those in PT and 1DRF groups. Among PT, 1DRF, and RL groups only RL hens showed significant increase of LHbeta mRNA synthesis when compared with control (without cLHRH-II). These results suggest that the responsiveness of the anterior pituitary to cLHRH-II increased in postmolt hens, indicating that the functions of this organ might be improved by induced molting.

Changes in the population of pituitary protein transcription factor-1 nuclei in the anterior pituitary during withdrawal and resumption of feeding in hens.

Pituitary protein transcription factor (Pit-1) is a member of a large family of protein transcription factors that include Pit-1, Oct-1, Oct-2, and Unc-86. The goal of this experiment was to determine whether the population of Pit-1-containing cells changes in the anterior pituitary of chicken by the regulation of feeding. White Leghorn hens were subjected to withdrawal and resumption of feeding. The anterior pituitaries were collected from hens at pretreatment, at 2 d after withdrawal of feeding (2DWF), and 1 d and 5 d after resumption of feeding (1DRF and 5DRF, respectively). Sections of the pituitaries were immunostained for Pit-1. They were examined under a light microscope with an image analysis computer system. The Pit-1 positive nuclei were found in the glandular cells in the cephalic and caudal lobes of the anterior pituitary in all four groups of hens. The Pit-1 cell population significantly increased in the 2DWF and 1DRF and decreased thereafter in 5DRF. These results suggests that feed withdrawal may stimulate Pit-1 expression in chicken, suggesting that Pit-1 may be involved in control of pituitary functions during the process of feed regulation.

Changes in the expression of TGFbeta-isoforms in the anterior pituitary during withdrawal and resumption of feeding in hens.

Our goal was to determine whether TGFbeta-isoforms were involved in the remodeling of the pituitary cell population which occurred by regulation of feeding. The current study examined whether TGFbeta-isoforms were produced in the anterior pituitary, and the mRNA expression of TGFbeta-isoforms changed during withdrawal and resumption of feeding. White Leghorn laying hens were subjected to feed withdrawal for 4 days with a resumption of feeding thereafter. The anterior pituitary tissues were collected from hens of pretreatment (PT), 3 days after feed withdrawal (3DFW), 1 and 5 days after the resumption of feeding (1DRF and 5DRF, respectively), and on the day of resumption of egg-laying (RL). They were processed for semi-quantification of TGFbeta2, beta3, and beta4 mRNA expressions by reverse transcription-polymerase chain reaction (RT-PCR) and for immunocytochemistry for TGFbeta3. TGFbeta2, TGFbeta3, and TGFbeta4 mRNA expression with a product size of 269, 236, and 163bp, respectively, was observed in the anterior pituitaries in all groups of hens. Although the expression of TGFbeta2 and TGFbeta4 mRNA did not show any significant change, that of TGFbeta3 mRNA significantly declined in the 1DRF hens and recovered by the resumption of laying. Immunostaining revealed that TGFbeta3 was located in the cytoplasm of glandular cells with granule forms in all groups of hens. The ovarian and oviductal weights sharply declined in the 3DFW and 1DRF groups, followed by a full recovery in the RL hens. These results indicate that TGFbeta2, beta3, and beta4 were expressed in the chicken anterior pituitary, and TGFbeta3 mRNA expression was changed in correlation with the regulation of feeding, suggesting that this isoform may play a significant role in the regulation of the glandular cell population and/or differentiation.

Changes in the population of immunoreactive S-100-positive folliculo-stellate cells in hens during induced molting.

The goal of this study was to determine whether the population of folliculo-stellate (FS) cells in the hen's pituitary change during induced molting. White Leghorn laying hens were subjected to induced molting by feed withdrawal; feeding was resumed on the fourth day after egg laying ceased. The anterior pituitaries were collected from hens at pretreatment, at 3 and 5 d after feed withdrawal and at 3 d after cessation of egg laying, 10 d after cessation of egg laying (6 d after resumption of feeding), on the day of and 1 wk after resumption of egg laying (RL and 1WRL, respectively). Pituitaries were processed to detect FS cells by immunocytochemistry for the S-100 protein. Sections were then examined under a light microscope with an image analysis computer system. S-100 immunoreactive cells were found in the cephalic and caudal lobes of the anterior pituitary in all groups of hens. The majority of S-100 immunoreactive cells formed clusters of cells that faced into the follicle and surrounded the glandular cells with long cytoplasmic processes. The S-100 immunoreactive area in the cephalic lobe was significantly increased in the RL group (P < 0.05), but decreased thereafter in 1WRL group. The S-100-positive cell area in the caudal lobe did not show significant changes during induced molting. These results suggest that FS cell population is likely to increase at the final stage of induced molting and may be involved in control of pituitary functions for the resumption of ovulation.

Cell proliferation and apoptosis in the anterior pituitary of chicken during inhibition and resumption of laying.

The goal of this study was to determine whether tissue rejuvenation of the anterior pituitary with cell proliferation and apoptosis occurs during inhibition and resumption of egg-laying. White Leghorn laying hens were subjected to inhibition of laying by feed withdrawal. Feeding was resumed on the fourth day of egg-laying cessation. All birds were injected ip with bromodeoxyuridine (BrdU) 1 h before tissue collection. The anterior pituitary glands were collected from hens of the following groups: pretreatment (PT), 3 and 5 days after starvation (3DS and 5DS, respectively), 3 days after cessation of laying (3DC), 10 days after cessation of laying (10DC, 6 days after resumption of feeding), and the day of and 1 week after resumption of laying (RL and 1WRL, respectively). They were processed for the detection of proliferating cells and apoptotic cells by BrdU immunostaining and terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end-labeling (TUNEL). Immunostaining for the anterior pituitary hormones was also conducted. In the cephalic lobe the BrdU-positive cells showed a higher frequency in RL than in PT, 3DC, and 1WRL. BrdU-positive cell frequency in the caudal lobe was greater in RL than in PT. TUNEL-positive cells in both cephalic and caudal lobes were increased markedly in the RL group. Their frequency in the cephalic lobe was greater in RL than in PT, and that in the caudal lobe of RL was higher than in any other group of birds. The areas of FSH-like cells in 10DC and RL were greater than those in PT to 3DC, and those of LH-like cells in RL were greater than those of 3DS to 3DC. PRL-like cells were decreased until 3DC and then gradually increased until 1WRL. GH-, TSH-, and ACTH-like cell areas showed tendencies to increase until 3DC, with decreasing thereafter. The sizes of FSH-like cells in 10DC to 1WRL and LH-like cells in RL were larger than those around cessation of laying. These results suggest that during inhibition and resumption of laying cell proliferation and apoptosis occur in the anterior pituitary tissue, which may cause a rejuvenation of tissue to improve the function of this organ.