Probiotic Bacillus subtilis C-3102 Improves Eggshell Quality after Forced Molting in Aged Laying Hens.

This study was carried out to evaluate the effects of probiotic Bacillus subtilis C-3102 feed additive on quality characteristics including strength, thickness, and weight of eggshells of Boris Brown laying hens. The control group (n=64) was fed a basal diet comprised of maize and feed rice, whereas the experimental group (n=64) was fed a basal diet supplemented with B. subtilis C-3102 (3×105 CFU/g) starting at 49 weeks of age. From 67 to 69 weeks, all hens were induced to molt using an anorexic program; then, the birds in both groups returned to their respective diets (from 69 to 82 weeks). Eggshell strength, measured six times with 60 eggs selected before the molting treatment, was significantly greater in the C-3102 group than in the control group at 51, 59, 63, and 66 weeks (3.45, 3.44, 3.28, and 3.13 kg/cm2; P<0.05, 0.05, 0.01, and 0.01, respectively). Moreover, eggshell strength-measured three times after the molting treatment-was significantly greater in the C-3102 group than in the control group at 73 and 77 weeks (3.79 and 3.65 kg/cm2; P<0.01 and 0.01, respectively). Eggshell thickness was also significantly greater in the C-3102 group than in the control group at 73 and 77 weeks (0.400 and 0.390 mm; P<0.01 and 0.01, respectively). Fecal samples collected from eight hens of each group at 70 weeks of age after forced molting, showed a significantly higher proportion of Lactobacillus spp. in the C-3102 group (8.94 log CFU/g) (P<0.05) than in the control group (8.63 log CFU/g). Clostridium spp. abundance was significantly lower in the C-3102 group (2.92 log CFU/g) than in the control group (4.3 log CFU/g). These results suggest that C-3102 supplementation improves eggshell quality in aged laying hens, particularly after forced molting.

Photoperiod-Specific Expression of Eyes Absent 3 Splice Variant in the Pars Tuberalis of the Japanese Quail.

The molecular mechanism underlying photoperiodic response in seasonal breeding animals such as the Japanese quail, red jungle fowl, sheep, mouse, and hamster involves thyroid-stimulating hormone beta subunit (TSHß) mRNA expression in the pars tuberalis stimulated by the extension in day length. Furthermore, this mechanism is regulated by eyes absent 3 (Eya3) in mammals. Even in birds, the expression of both TSHß and EYA3 is induced in the pars tuberalis by the extension in day length; however, the relationship between the two genes is unknown. To clarify the function of EYA3 in quail photoperiodism, in the present study, we performed mRNA structure analysis of the Japanese quail EYA3 mRNA using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. The results revealed that there are four types of splice variants within regions of exons 7, 8, and 9 of quail EYA3 mRNA. Among the four splice variants of quail EYA3, the splice variant containing exon 7 was expressed in the pars tuberalis on the first long day, when quails were transferred from the short-day condition to the long-day condition. The results indicate that EYA3 splice variant containing exon 7 is involved in the photoperiodic response of the pars tuberalis in the Japanese quail.

Expression of Prolactin Receptor mRNA in Lactotrophs and Somatotrophs of the Chicken Anterior Pituitary Gland.

Prolactin (PRL) is a hormone mainly secreted by the anterior pituitary gland. In birds, PRL exerts a variety of physiological functions in target tissues expressing the PRL receptor (PRLR). In chicken, the PRLR mRNA is abundant in the anterior pituitary gland, but its regional and cellular localization are unknown. In the present study, we investigated the expression of the PRLR mRNA in cephalic and caudal lobes of the chicken anterior pituitary gland. Real-time polymerase chain reaction (PCR) revealed high levels of PRLR mRNA in both cephalic and caudal lobes. In situ hybridization revealed that the PRLR mRNA was distributed in a wide area of both lobes, and co-localized with the PRL and growth hormone (GH) mRNAs in the cephalic and caudal lobes, respectively. These results suggest that PRL exerts autocrine/paracrine effects through PRLR on PRL-producing lactotrophs and GH-producing somatotrophs in the chicken anterior pituitary gland.

Two chicken neuromedin U receptors: characterization of primary structure, biological activity and tissue distribution.

Neuromedin U (NMU) is a bioactive peptide that is involved in a variety of physiological functions. Two of its receptors, NMUR1 and NMUR2, have been identified and characterized in mammals. In this study, we performed cDNA cloning of chicken NMUR1 and NMUR2, and characterized their primary structure, biological activity, and expression patterns in chicken tissues. The chicken NMUR1 and NMUR2 cDNAs encoded 438 and 395 amino acid sequences, respectively. Chicken NMUR1 showed 54.8%-56.5% sequence identity with human, rat, and mouse NMUR1, and NMUR2 shared 67.3%-70.1% sequence identity with mammalian orthologs. Both chicken receptors have typical characteristics of G-protein-coupled receptors with seven transmembrane domains and the D/ERY motif. An increase in intracellular Ca(2+) mobilization was observed in HEK293 cells transfected with chicken NMUR1 or NMUR2 cDNA and treated with chicken or rat NMU. Real-time PCR analysis revealed that NMUR1 mRNA was preferentially expressed in the intestinal tissues such as the duodenum, jejunum, ileum, cecum, and colon/rectum, and brain regions such as the midbrain and optic lobe, and the ovary in adult hens. NMUR2 mRNA was exclusively expressed in the brain regions such as the cerebrum and midbrain. These results indicate that NMUR1 and NMUR2 mRNAs, which encode functional receptor proteins, are expressed in chicken tissues with different distribution patterns.

Molecular characterization of structure and tissue distribution of chicken neurotensin receptor.

Neurotensin, a tridecapeptide, is distributed in a wide range of tissues and exhibits multiple functions through its receptors. Hitherto molecular characterization of the neurotensin receptor has been reported in mammalian, amphibian, and fish species but not in avian species. In this study, we cloned the cDNA encoding chicken neurotensin receptor from the duodenum and characterized its primary structure, biological activity and distribution in the gastrointestinal tract. The cDNA encoded a protein consisting of 399 amino acids that had significant overall sequence homology to other vertebrate neurotensin receptor 1 with higher extent in the seven transmembrane domains. Chicken neurotensin increased intracellular Ca(2+) concentrations in human embryonic kidney 293 cells transiently expressing the chicken neurotensin receptor 1. Real-time PCR analysis showed that chicken neurotensin receptor 1 mRNA is expressed throughout the gastrointestinal tract with markedly higher level in the colon/rectum. These results indicate that the chicken neurotensin receptor 1 is involved in gastrointestinal functions through an intracellular signaling pathway accompanied by an increase in Ca(2+) levels.

Molecular characterization of sequence and expression of chicken GPR39.

GPR39 has been recently proposed to be a specific receptor for a novel anorexic peptide, obestatin, isolated from rat stomach. Obestatin is generated from the proprotein for ghrelin by proteolytic cleavage and shows opposing action to ghrelin in the regulation of food intake and gastrointestinal movement. In this study, we performed cDNA cloning for chicken GPR39 and characterized expression profiles of its mRNA in chicken tissues. Chicken GPR39 cDNA encoding 462 amino acids was cloned from chick duodenum. The amino acid sequence showed high homology to human (62.6%), mouse (62.6%), and rat (65.3%) GPR39. A computer-assisted search for chicken GPR39 cDNA sequence in the chicken genome database revealed that chicken GPR39 gene consists of two exons separated by an intron. Real-time PCR analysis revealed the expression of GPR39 mRNA in a wide range of tissues with the highest level in the duodenum in chicks and hens. The expression level in the duodenum rapidly increased during the early post-hatch period. Interestingly, relatively higher expression was observed in the oviduct, vagina and uterus in hen. These findings suggest that GPR39 is involved in the regulation of gastrointestinal and reproductive functions in chicken.

Molecular characterization of chicken growth hormone secretagogue receptor gene.

Synthetic growth hormone secretagogues stimulate growth hormone secretion by binding to a specific receptor, growth hormone secretagogue receptor (GHS-R). In this study, we investigated the cDNA and the genomic structure of chicken GHS-R. Chicken GHS-R gene is composed of two exons separated by an intron. Two GHS-R mRNA species, cGHS-R1a and cGHS-R1a-variant (cGHS-R1aV) are generated by alternative splicing of a primary transcript. cGHS-R1a protein is predicted to have seven transmembrane domains by a high degree of amino acid sequence identity with mammalian and teleost homologs. cGHS-R1aV lacks the transmembrane-6 domain due to a 48 bp deletion. RT-PCR analysis showed widespread tissue distributions of cGHS-R1a and cGHS-R1aV mRNAs with much higher amounts of cGHS-R1a in all the tissues.