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White Leghorn chickens were divided into the control, low-carbohydrate (CHO), and CHO-free groups to investigate dietary CHO’s significance on histological features of chicken ileal mucosa. Paraffin sections of distal ileum from each chicken were stained by periodic acid-Schiff reaction and subjected to morphometrical analysis. Most villi in the control group had a fingerlike shape but those of the experimental groups showed irregular shapes. Villus height, crypt depth and the number of mitotic cells per crypt were significantly lower in the CHO-free group than in the control group. The density of goblet cells also showed a significant decreasing trend with a reduction in dietary CHO level. In conclusion, dietary CHO positively affects the proliferation of epithelial cells in the chicken ileum.

Typical skeletal muscles are composed of mixed muscle fiber types, which are classified as slow-twitch (type I) and fast-twitch (type II) fibers, whereas pectoralis major muscles (PMs) in broiler chickens are 100% composed of type IIb fast-twitch fibers. Since metabolic properties differ among muscle fiber types, the combination of muscle fiber types is involved in physiological functions and pathological conditions in skeletal muscles. In this study, using serial block-face scanning electron microscopy, we compared three-dimensional (3D) mitochondrial properties in type IIb fibers in broiler PMs and those in type I fibers of broiler gastrocnemius muscles (GMs) heterogeneously composed of slow- and fast-twitch muscle fibers. In type I fibers in the GMs, elongated mitochondria with numerous interconnections to form a substantial network among myofibrils were observed. Along with lipid droplets sandwiched by mitochondria, these features are an adaptation to effective oxidative respiration and constant oxidative damage in slow-twitch muscle fibers. In contrast, type IIb fibers in the PMs showed small and ellipsoid-shaped mitochondria with few interconnections and no lipid droplets, forming a sparse network. The mitochondrial spatial network comprises of active mitochondrial dynamics to reduce mitochondrial damage; therefore, type IIb fibers possess physiologically low capacity to maintain mitochondrial wellness due to static mitochondrial dynamics. Based on 3D mitochondrial properties, we discussed the contrasting physiological functions between type I and IIb fibers and proposed a high contractile power and low stress resistance as unique physiological properties of broiler PMs.

Neurotensin is secreted from intestinal N cells in response to the food ingestion. Influences of different dietary protein levels on neurotensin-immunoreactive cells in the chicken ileum were examined by using immunohistochemical and morphometrical techniques. The results showed that dietary protein had an obvious influence on neurotensin-immunoreactive cells in the chicken ileum. Four experimental groups were used, with dietary crude protein (CP) levels of 18% (control), 9%, 4.5% and 0%. Enteroendocrine cells showing neurotensin-immunoreactivity were located in crypts and villous epithelium in all groups. Most of the neurotensin-immunoreactive cells in the villous epithelium showed pyramidal or spindle-like shape with a long cytoplasmic process reaching the intestinal lumen, but cells with round or oval shape were found in the CP4.5% and 0% groups. Frequencies of occurrence of neurotensin-immunoreactive cells in the CP18%, 9%, 4.5% and 0% groups were 42.4±3.3, 36.6±2.2, 30.8±2.6 and 25.4±3.8 (cell count per mucosal area: cells/mm2, mean±SD), respectively. There were significant differences in neurotensin-immunoreactive cell frequency between the control and lower CP level, 4.5% and 0%, groups. A significant correlation was found between frequency of occurrence of neurotensin-immunoreactive cells and daily protein intake. These results indicate that ingested protein is likely to be a potential signal for neurotensin production and secretion of N cells in the chicken ileum.

The nasal mucosa plays an important role in the immune system, with nasal mucous cells secreting mucin that, along with pili, exclude foreign substances from intervening. Nasal mucosal-associated lymphoid tissue (NALT), present in the nasal lamina propria, acts as a local immune system. In birds, the Harderian gland in the orbit also plays an important role in the local immune system. In this study, we analyzed the pathway from the nasolacrimal duct to the nasal cavity in chickens and the distribution of the nasal mucous cells responsible for defense mechanisms against pathogens. To determine the three-dimensional structure of the pathway from the nasolacrimal duct to the nasal cavity, we made casts of the anatomy by injecting an acrylic resin into the area. We then prepared paraffin sections to determine the distribution of the NALT and mucous cells. The mucous gland was clearly seen in the mucosal epithelium of the nasal cavity, suggesting that the pathway along the nasal cavity develops a nonspecific immune system to deal with large foreign substances, such as bacteria, using mucins that are secreted from the mucous glands. Hence, there is not only a physical barrier but also an antibacterial activity. Unlike in other animals, morphologically, the nasolacrimal duct in chicken becomes the ventral nasal meatus and opens into the choanae in the caudal portion of the nasal cavity. NALT was prominently present in the lamina propria of the ventral nasal meatus, suggesting the presence of a specific immune system protecting against avian viruses. Thus, responses to vaccine stimulation could be developed from tissues along the pathway of the ventral nasal meatus via the nasolacrimal duct running from the punctum. These morphological studies suggest that the instillation of eye drops could be used as an efficient vaccination method for avoiding respiratory diseases.

Glucagon-like peptides (GLPs) are secreted from intestinal L cells and stimulate various physiological functions in the gastrointestinal tract. The secretion of GLPs is influenced by macronutrient ingestion. This study aims to clarify the effects of dietary carbohydrate (CHO) on L cells in the chicken ileum. Six-week-old, male White Leghorn chickens were divided into three groups: control, low-CHO and CHO-free, with five chickens in each group. Paraffin sections were made from the proximal and distal ileum of each animal and subjected to immunohistochemistry for GLP-1 and GLP-2 peptides and in situ hybridization for proglucagon (PG) mRNA. A significant reduction of GLP-1- and GLP-2-immunoreactive cells was observed in the two experimental groups compared with that in the control. A reduction of cells expressing PG mRNA was observed in the proximal and distal ileum of the CHO-free group compared with that in the control. The ratio of GLP-1-immunoreactive cells showing Ki-67 immunoreactivity was significantly lower in the distal ileum of the CHO-free group than that in the control group. These data suggest that dietary CHO is an effective stimulator for modifying L cell density in the chicken ileum.

Glucagon-like peptide (GLP)-1 colocalizes with neurotensin (NT) in the same enteroendocrine cells (EECs) of the chicken ileum. The present study was designed to clarify the influence of dietary carbohydrate (CHO) on the colocalization pattern of GLP-1 with NT in the chicken distal ileum. Male White Leghorn chickens at 6 weeks of age (n=15) were divided into three groups, a control and two experimental (low-CHO and CHO-free), with five chickens in each, and fed control or experimental diets for 7 d. Distal ileum was collected from each bird as a tissue sample and subjected to double immunofluorescence staining to detect GLP-1 and NT. Three types of EEC, GLP-1+/NT+, GLP-1+/NT− and GLP-1−/NT+, were demonstrated in the chicken ileum. GLP-1+/NT+ cells in the control group had a spindle-like shape with a long cytoplasmic process, but those in the experimental groups were round and lacked a cytoplasmic process. The ratio of GLP-1+/NT+ cells was significantly decreased in the two experimental groups compared with that in the control group. The ratio of GLP-1+/NT+ cells was significantly lower than those of GLP-1+/NT− and GLP-1−/NT+ cells in the two experimental groups. Most cells that were immunoreactive for GLP-1 and NT antisera lacked signals of proglucagon (PG) and NT precursor (NTP) mRNA in the experimental groups. The number of EECs expressing PG and NTP mRNA signals showed tendencies for decreases with a reduction of dietary CHO level. These findings suggest that dietary CHO could be a significant regulator of the pattern of colocalization pattern of GLP-1 with NT in the chicken ileum.

This study was designed to reveal the role of posttransportation grazing on the physiological condition and meat quality traits of Black Bengal goats. Twenty-four castrated male Black Bengal goats were divided into a control (untransported) group and three treated groups: Walking and Transportation with Human Interference group (WTHI) (30 min walking before 6 hr transport and then 30 min walking with human interference), posttransportation grazing for 48 hr (PTG1), and posttransportation grazing for 72 hr (PTG2). The WTHI and PTG1 groups had a significant reduction in their blood concentrations of tri-iodothyronine (T3 ) and thyroxine (T4 ), and a significantly higher neutrophil and lymphocyte (N:L) ratio compared with those of the control group. Blood concentrations of T3 and T4 and the N:L ratio of PTG2 returned to the control level after 72 hr of grazing. The final pH and water-holding capacity values of meat were significantly higher in the WTHI group than in the control group, but those in the PTG2 group returned to the control level after 72 hr of grazing. These results demonstrate that posttransportation grazing for 72 hr is effective for recovering from damage induced by transportation stress.

Glucagon-like peptide (GLP)-1 and neurotensin (NT) are distributed throughout the chicken ileum. Here, we attempt to determine if GLP-1 and NT co-localize in the chicken ileum by using immunofluorescence, immunocytochemistry and in situ hybridization techniques. Three types of enteroendocrine cells, GLP-1+/NT+, GLP-1+/NT− and GLP-1−/NT+ cells, were detected in the mucosal epithelium by the double immunofluorescence method. The ratio of GLP-1+/NT+ cells at the crypts in the distal ileum was significantly higher than that in the proximal ileum. The ratios of the three cell types were similar along the crypt–villous axis in the proximal ileum but the percentage of GLP-1+/NT+ cells significantly decreased at the middle part of villi relative to crypts and the bottom part of villi in the distal ileum. Enteroendocrine cells that were immunoreactive to both GLP-1 and NT peptides and showed both proglucagon and NT precursor mRNA signals were found in the crypts of the distal ileum but not in the villous epithelium. The results from performing an immunocytochemical method with colloidal gold indicated that the GLP-1 content within GLP-1+/NT+ cell secretory granules decreased stepwise from the crypt to the middle part of the villus but the NT content in these granules increased in this direction. These findings reveal that the cells producing both GLP-1 and NT are mainly localized in the crypts of the chicken ileum but these endocrine cells specialize in NT-producing cells at the villous epithelium of the distal ileum.

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

An antigen retrieval method for immunohistochemical staining of glucagon-like peptide (GLP)-2-immunoreactive cells was investigated in the chicken small intestine. GLP-2-immunoreactive cells were observed as open-typed endocrine cells in the villous epithelium and crypts on both antigen retrieval agent-treated and untreated preparations. No obvious differences were detected in morphological features of GLP-2-immunoreactive cells between treated and untreated preparations. The frequencies of occurrence of GLP-2-immunoreactive cells, however, were significantly different in treated and untreated preparations: in the proximal and distal regions of jejunum and ileum obtained from untreated preparations, the frequencies of occurrence were 0.5 +/- 0.2, 0.7 +/- 0.1, 0.9 +/- 0.2 and 1.5 +/- 0.3, respectively (cell numbers per mucosal area: cells/mm(2), mean +/- SD), whereas those from treated sections were 14.7 +/- 2.3, 19.8 +/- 2.3, 23.5 +/- 4.7 and 34.6 +/- 4.9 cells/mm(2), respectively. These data indicate that this antigen retrieval method is able to make immunoreactive GLP-2 available for detection and that GLP-2 may act as one of the common hormones secreted by L cells in the chicken small intestine., Article, JOURNAL OF VETERINARY MEDICAL SCIENCE. 76(4):565-568 (2014)

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P

The purpose of the present study was to investigate the possibility of immunoelectron microscopic observation of endocrine cells in paraffin-embedded tissues. The procedure, which involves reprocessing from sliced tissues and immunohistochemical staining by colloidal-gold immunolabeling of paraffin sections from paraffin blocks, was able to reveal the fine characteristics of secretory granules containing glucagon-like peptide-1. Morphometric analyses of the secretory granules showed no significant differences between the reprocessing procedure and a conventional post-embedding procedure, which was performed as a control. The reprocessing procedure has some advantages besides providing information on secretory granules containing the amino acid peptide. For example, the same cell can be observed under both a light microscope and the electron microscope. In addition, the high-electron densities of silver-enhanced gold particles are easily recognized, and the boundary between the profile of the granules and the immunogold labeling is clearly shown at the electron microscopic level. Furthermore, the procedure, which is inexpensive and does not require special devices, can effectively use precious samples that are already paraffin-embedded and unable to be obtained twice, such as the case for endangered animals and rare pathological tissues. To the best of our knowledge, the present study is the first to report the advantages of the reprocessing method for sliced paraffin sections of gut endocrine cells., Article, JOURNAL OF VETERINARY MEDICAL SCIENCE. 76(3):389-394 (2014)

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small-diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large-diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small- and large-diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface-to-surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center-to-center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.

Somatostatin is well known as an inhibitor of growth hormone release from the anterior pituitary. Its effects are exerted via 5 subtypes of receptors, which are named SSTR1 through 5. We recently reported that intracerebroventricular (ICV) injection of somatostatin stimulates feeding behavior in chicks. However, the specific receptors which mediate this orexigenic effect have not been identified in chicks. Thus, the purpose of the present study was to identify the receptor subtypes involved in somatostatin-induced feeding using 5 somatostatin analogs. Chicks that received vapreotide and octreotide (less than 3nmol), which are agonist of SSTR2 and SSTR5, increased their food intake. Additionally, chicks ICV injected with BIM23056 or L-817,818 (SSTR3 and SSTR5 agonists, respectively) also had increased food intake. However, ICV injection of the SSTR4 agonist L-803,087 did not cause an orexigenic effect, suggesting that SSTR4 might not be important in somatostatin-induced feeding behavior. In summary, results from this study may be interpreted as SSTR2, SSTR3 and SSTR5 are related to somatostatin-associated feeding behavior in chicks.

The influence of varying dietary proteins levels from 0% to 18%, which is the level of dietary protein requirement, on plasma IGF-I concentration and tissue IGFBP-2 and -4 gene expression was examined. Body weight gain was gradually lowered by decreasing dietary protein levels from 18% to 4.5%, and body weight was lost when chickens fed a protein-free diet. Plasma IGF-I concentration of chickens fed the 18% crude protein diet was the highest of all, and it was gradually decreased by reducing dietary protein concentrations. Gene expression of IGFBP-2 in the gizzard was elevated by decreasing dietary protein levels from 18% to 9%, and the further reduction in dietary protein content lowered gizzard IGFBP-2 gene expression. No significant change in hepatic IGFBP-4 gene expression was observed in chickens fed experimental diets with varying dietary protein levels. Multiple linear regression analysis revealed that the influence of the change in plasma IGF-I concentration and gizzard IGFBP-2 gene expression on body weight change of chickens fed varying amounts of dietary protein seemed to be independent because the interactive effect of both factors on body weight change was not significant.

The purpose of the present study was to determine if central administration of somatostatin influences feeding behavior in layer chicks. Five- to 7-day-old chicks that received intracerebroventricular (ICV) injections of 0.5 or 2 nmol somatostatin increased their food intake at 30 and 60 min after the injection, suggesting that central somatostatin serves as an orexigenic neuropeptide in chicks. This hypothesis was further supported since chicks ICV injected with 0.5 or 2 nmol cortistatin, which binds to somatostatin receptors, also had increased food intake at the same time. Somatostatin-associated feeding behavior was attenuated by co-administration of 20 nmol beta-funaltrexamine (an opioidergic mu-receptor antagonist) (to 31% of the orexigenic effect of somatostatin at 60 min after the injection) but not ICI-174,864 or nor-binaltorphimine (antagonists of opioidergic delta- and kappa-receptors, respectively). Co-administration of 13 nmol yohimbine, an adrenergic alpha-2 receptor antagonist, also attenuated the orexigenic effect of somatostatin (to 31% of the orexigenic effect of somatostatin at 60 min after the injection). These results suggest that somatostatin-associated feeding behavior is mediated by opioidergic mu- and adrenergic alpha-2-receptors in chicks.

In previous studies, two types of offspring were generated from germline chimera between NH-413 strain (donor) and White Leghorn L-M strain (recipient). Phenotype and symptom of type-I offspring were quite similar to that of the NH-413 strain. In the other offspring of type-II, feather color showed mixture of white and brown and the symptom was not dominantly indicated. In the present studies, sexually matured males and females of the type-l were mated each other. Form these mating. chickens manifesting completely same phenotype to that of the donor NH-413 strain; brown feather color and symptoms of Muscular dystrophy. were regenerated. Therefore, complete regeneration of the muscular dystrophy chickens Could be achieved by mating males and female offspring derived from the germline chimeras. Fertility, hatchability and Survival rate of these regenerated offspring were significantly improved as compared to that of the original NH-413 strain. The established strategies should be one of the useful systems to regenerate chickens with muscular dystrophy., Article, JOURNAL OF POULTRY SCIENCE. 46(2): 123-126(2009)

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of secretin/glucagon/VIP family, has a potent action oil the pancreatic secretion in mammals. The present study aimed to clarify the distribution of neural elements containing PACAP27 or 38 in the chicken pancreas. These two peptides were detected in the neural elements and showed the similar distributional pattern in the chicken pancreas. Intrapancreatic ganglia containing PACAPs-immunoreactive cells were found in the interlobular connective tissue. Nerve fibers showing immunoreactivity for PACAPs made the dense network around arterioles and were distributed in lamina propria of secretory ducts and the exocrine pancreas. Double labeling immunohistochemistry for PACAPs and islet hormones revealed that PACAPs-immunoreactive nerve fibers were ill contact with B and D cells in B islets, but rarely distributed in A islets. These data suggest that PACAPs have relation to the regulation of the secretion from exocrine tissue and B and D cells in B islets of the chicken pancreas., Article, JOURNAL OF POULTRY SCIENCE. 46(3): 234-239(2009)

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12×3×0.25mm, ∼0.1g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120 s (∼7.2Gy) at stages 13 to 17 showed a hatchability of 35% (106/301), whereas the hatchability of unshielded embryos was 26% (27/105). The relative population of gonadal PGCs at stage 30 for embryos irradiated at stage 13 with or without shielding was 13 and 5%, respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or without shielding and transfused with quail embryonic blood containing PGCs each exhibited ∼130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to ∼7.2Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras.

Effect of pear procyanidins on ethanol-induced gastric ulcer in rats was investigated. In a preliminary experiment, the percentage of lesion area to total gastric surface area (ulcer index) increased with increases in ethanol concentration (40–80%) and the length of time after ethanol treatment (60–120 min). Migration of leukocytes, estimated by myeloperoxidase activity in mucosa, was involved in the occurrence of an ulcer, whereas expansion of the ulcer seemed to be due to leukocytes that had already migrated. Highly polymerized procyanidins extracted from ‘Winter Nelis’ pear fruit, orally administered (20 mg/rat) before 60% ethanol treatment, exhibited a high level of antiulcer capacity whereas chlorogenic acid alone seemed to have a negative effect. The trend of myeloperoxidase activity was similar to the trend of the ulcer index. A mixture of those polyphenols had a significant protective effect. The results suggest that the antiulcer effect of pear procyanidins may be due to their strong antioxidant activity.

The dynamics of L cells along the crypt-villous axis were investigated in the ileum of male White Leghorn chicks (7 d of age, n = 5). Immunohistochemistry was used to detect the expression of glucagon-like peptide (GLP)-1 and an in situ hybridization technique to detect proglucagon messenger RNA (mRNA). Immunocytochemistry using colloidal gold was also applied to quantitatively evaluate the GLP-1 content. The cells expressing a proglucagon mRNA signal were distributed mainly in the crypts and the bottom of the villi but were never found in the upper part of the villi. Most of the cells expressing a proglucagon mRNA signal (97%) were immunoreactive for GLP-1 antiserum. In contrast, GLP-1 immunoreactive cells were distributed from the crypts to the middle part of the villi, and only 55% of them expressed a proglucagon mRNA signal. Quantitative evaluation by immunocytochemistry of GLP-1 using colloidal gold revealed that the GLP-1 content was significantly lower in L cells located in the villous epithelium than that of L cells located in the crypts (P < 0.01). These findings indicate that L cells in the chicken ileum mature and complete GLP-1 production in the crypts. L cells in the villous epithelium secrete GLP-1 but do not synthesize this peptide.

To evaluate the health benefits of Chinese quince and quince phenolics, their antioxidant properties and antiulcerative activity were investigated in comparison with apple phenolics as a reference. The strength of antioxidant activity and DPPH radical scavenging activity of these fruit phenolics varied according to different in vitro evaluation systems, whereas the antioxidative property of rat blood increased in all rats orally administered phenolics. Ferulic acid and isoferulic acid were detected as major metabolites in rats given apple phenolics, quince phenolics, and 5-caffeoylquinic acid standard. (-)-Epicatechin and its 3'-O-methyl ether could be detected in rats administered apple phenolics and (-)-epicatechin standard. In the ethanol-induced gastric ulcer, pre-administration of Chinese quince and quince phenolics suppressed the occurrence of gastric lesions in rats, whereas apple phenolics seemed to promote ulceration. The trend of myeloperoxidase activity was similar to that of the ulcer index. The results showed that Chinese quince and quince phenolics might have health benefits by acting both in blood vessels and on the gastrointestinal tract.

The chicken Harderian gland (HG) was investigated using single immunohistochemical staining for one of the three different immunoglobulins (Igs) followed by Alcian blue/periodic acid Schiff (AB/PAS) staining and triple immunohistochemical staining for all of the Igs with hot water treatment. In the HG of 5-week-old chickens, IgG-containing plasma cells were more frequent than IgA- and IgM-containing cells. These numerous IgG-containing cells were predominantly accumulated in the central region of the stroma, whereas a small number of IgA- and IgM-containing cells were scattered in the peripheral region of the stroma. Also, the plasma cells containing PAS-positive Russell bodies (RBs) exhibited distinct immunoreactivity for one of the Igs, being inversely proportional to the intensity of PAS reaction. The RB-containing cells positive for IgA were more frequent than those positive for IgM, whereas those positive for IgG were very rare. The most distinct feature of the IgG-containing plasma cells was a PAS-positive globule located close to the nucleus. Triple immunostaining with hot water treatment simultaneously identified these three Igs in normal plasma cells and RB-containing ones in the stroma of the chicken HG.

The distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurones was investigated in the chicken caecum. Double staining combined NADPHd histochemistry with immunohistochemistry for neural nitric oxide synthase (nNOS) indicated that NADPHd-positive neurones also showed immunoreactivity for nNOS. NADPHd-positive nerve cell bodies were observed in both the myenteric and the submucous plexuses. Nerve fibres showing enzyme activity were mainly distributed in the circular muscle layer, but only a few fibres in the mucosal layer. Fine nerve fibres showing NADPHd activity were found running between germinal centres in the caecal tonsil. Quantitative analysis showed no significant differences in the number of enzyme-positive nerve cell bodies per ganglion of the myenteric and the submucous plexuses among three different caecal regions; proximal, middle and distal regions. Larger numbers of ganglia were detected in the submucous plexus than the myenteric plexus at all three regions. These data indicate that nitrergic neurones in the submucous plexus mainly project to the circular muscle layer in the chicken caecum. It is possible that nitrergic nerves regulate the motility of the chicken caecum.

The fine structure and immunocytochemical localization of serotonin in the cells of the receptor line were studied in the parietal eye and pineal organ proper of the Japanese grass lizard, Takydromus tachydromoides. Typical photoreceptor cells (PC) were the predominant cell type in the receptor line of the parietal eye, the outer segments of which had regular stacks of numerous disks similar to those of cones. The pineal organ contained relatively few PCs, which showed less well-developed outer segments than those of the parietal eye. In contrast, secretory rudimentary photoreceptor cells (SRPC) accounted for the majority of receptor cells in the pineal organ. These cells were structurally characterized by whorl-like lamellar outer segments and numerous dense-cored vesicles (80-280 nm in diameter). A small number of SRPC were also found in the parietal retina, which were similar to those in the pineal organ. In the parietal-pineal complex, numerous mitochondria located in the PC were larger and rounder than those in the SRPC. In the PC, basal processes prossessed only synaptic ribbons, whereas in the SRPC some of these processes contained synaptic ribbons and others contained dense-cored vesicles, rarely having both. Serotonin-immunoreactive cells were found not only in the pineal organ but also in the parietal eye, which closely resembled the cells of the receptor line in their size and shape. Furthermore, on immunoelectron microscopy for serotonin using the protein A-gold technique, gold particles indicating serotonin-immunoreactive sites were restricted in the core of dense-cored vesicles in the SRPC of the pineal organ. Regional differences in the distributions of the PC, SRPC and serotonin-immunoreactivity were found in the parietal-pineal complex.

Localization of neuropeptides (galanin, PACAP 27 and 38) was investigated in the chicken Harderian gland by immunohistochemistry. Immunoreactivity for three peptides examined in this study was detected on nerve elements in the pterygopalatine ganglion and the gland. Pterygopalatine ganglion contained nerve cell bodies showing immunoreactivity for galanin, PACAP 27 and 38. Dot-form immunoreactivity for PACAPs was also detected around immunonegative nerve cell bodies. Galanin immunoreactivity was observed on larger nerve cell bodies and those were most commonly observed in the ganglion. Nerve fibers showing immunoreactivity for the neuropeptides were distributed in the interstitial tissue of the Harderian gland. Fine immunoreactive nerve fibers were found running beneath the capsule and between acini. No im-munoreactive nerve fibers were found contacting with epithelial cells of the collecting duct. The present data suggested that nerves containing galanin and PACAPs have a relation to the regulation of the immune function of the chicken Harderian gland.

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.

Colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in L-cells was investigated in the chicken ileum by using double immunofluorescent and immunocytochemical techniques. Ultrastructural features of L-cells were also clarified in this study. L-cells showing immunoreactivity for both GLP-1 and GLP-2 were distributed in the whole ileum. They showed comma-like or flask-like shape and were located in epithelium of crypts and lower part of intestinal villi. L-cells showing GLP-1-immunoreactivity only were found in epithelium of lower and middle parts of intestinal villi. Transmission electron microscopy indicated that L-cells identified by colloidal gold-labeled immunocytochemistry were covered apically with microvilli, open-type and contained many secretory granules in their perikarya. These secretory granules without halo were round to oval in shape and showed moderate electron density. The longest and shortest diameters of secretory granules were 355 +/- 62 nm (mean +/- SD) and 287 +/- 48 nm, respectively. Double labeling immunocytochemistry using two different sizes of particles (6 and 12 nm in diameter) of colloidal gold revealed that GLP-1 colocalized with GLP-2 in the same secretory granules. This study advances new morphological data about the endocrine system of the chicken small intestine., Article, JOURNAL OF VETERINARY MEDICAL SCIENCE. 75(10):1335-1339 (2013)