Yukinori Yoshimura

Expression of Avian β-Defensins in the Oviduct and Effects of Lipopolysaccharide on Their Expression in the Vagina of Hens

The aims of this study were to (i) determine the types of avian beta-defensin genes (AvbetaD) expressed in the hen oviduct and (ii) to examine the effects of lipopolysaccharide (LPS) treatment in vivo on their expression in the vagina. Birds were i.v. treated with LPS (1 mg/kg of BW), and subsequently the oviducts were analyzed 0, 3, 6, 12, or 24 h after LPS administration. The mRNA expression for AvbetaD was examined by reverse transcription-PCR using RNA preparations from the mucosal tissues of all the oviductal segments. Furthermore, changes in their mRNA expression profiles in the vagina were analyzed by semiquantitative reverse transcription-PCR. The AvbetaD-1, -2, -3, -4, -5, -7, -8, -9, -10, -11, and -12 were identified in each oviductal segment from infundibulum to vagina. Among these AvbetaD, the expression of AvbetaD-3, -5, -10, -11, and -12 in the vagina were significantly increased in response to LPS treatment, whereas the others did not show significant changes. These results suggest that all 11 types of AvbetaD are expressed in the hen oviduct and at least 5 of them in the vagina show increased expression in response to LPS.

Expression of Toll-like receptors (TLRs) and TLR4 response to lipopolysaccharide in hen oviduct.

The aim of this study was to determine the types of Toll-like receptors (TLRs) expressed in the hen oviduct, and to confirm that vaginal tissue expressing TLR4 responds to lipopolysaccharide (LPS). Healthy laying hens were intravenously or intravaginally injected with LPS, PBS or untreated. The expression pattern of TLRs in the whole oviduct and the effects of LPS on TLR4 and IL-1beta in the vagina were examined by semi-quantitative RT-PCR. The population of cells containing TLR4 protein was examined by immunohistochemistry. The expression of 6 types of TLRs (TLR1 type 2, TLR2, TLR3, TLR4, TLR5 and TLR7) were identified in all segments of the oviduct. The densities of PCR products for each TLR showed a tendency to be greater in the vagina than in the magnum and isthmus. Immunoreactive TLR4 was localized in the epithelial cells and leukocytes in the isthmus, uterus and vagina. Intravenous injection with LPS increased the TLR4 expression and the population of TLR4-immunopositive cells in the vagina. Intravaginal injection with LPS resulted in the increase of leukocytes in the mucosal tissues in association with an increase of TLR4 expression and TLR4-immunopositive cells in the vagina. The IL-1beta expression was also enhanced in a similar manner to that of TLR4. These results suggest that the hen oviduct expresses at least 6 types of TLRs including TLR4 with a greater expression in the vagina. Vaginal tissue expressing TLR4 responds to LPS and in turn upregulate cellular functions to synthesize cytokines. Such expression and functions of TLRs may play an essential role in oviductal innate immunity for host defense.

Immunocytochemical study of cell proliferation in the cystic ovarian follicles in cows.

We examined the frequency of proliferating cells in cystic, atretic and healthy antral follicles to determine whether a disorder of cell proliferation was responsible for the occurrence of bovine cystic follicles. Paraffin sections of healthy follicles and various stages of atretic and cystic follicles were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). The PCNA-positive cells were counted in 4 different regions of a follicle from the apical to the basal side. In the granulosa layer, a significantly higher frequency of PCNA-positive cells was observed in the healthy follicle in the basal region as compared with the apical region. A similar pattern of PCNA-positive cells population was observed in the granulosa layer of atretic follicles, although the frequency in the basal region was significantly lower in the atretic than the healthy follicle. The rate of cell proliferation in the granulosa layer of cystic follicles was markedly lower at the basal region than that of atretic follicles. In the theca interna, the frequency of PCNA-positive cells in atretic follicles at the early stages was higher than that in cystic follicles at the early stages. These results suggest that in the healthy follicle the proliferative activity of granulosa cells is higher in the basal than the apical region, and that the cell proliferation activity in the granulosa and theca interna may decrease in association with the induction of a follicular cyst.

Poultry production profile and expected future projection in Bangladesh

The aim of this paper is to review the current status of poultry production and its future prospects in Bangladesh, covering both rural and commercial poultry production. As an important sub-sector of livestock production, the poultry industry in Bangladesh plays a crucial role in economic growth and simultaneously creates numerous employment opportunities. The poultry industry, as a fundamental part of animal production, is committed to supplying the nation with a cheap source of good quality nutritious animal protein in terms of meat and eggs. Two main systems of poultry production are common in Bangladesh nowadays: commercial poultry production – where birds are kept in total confinement, and traditional scavenging or semi-scavenging poultry production. Approximately 20% of the protein consumed in Bangladesh originates from poultry. With the exception the dip in production due to the recent Avian Influenza outbreak, the growth of this industry in terms of standards of commercialization, is very rapid. A gap still exists between the requirement and supply of poultry meat and eggs within the recent frame-work of the informal marketing system that is currently used. Among poultry species, the chicken population is dominant over others, at almost 90%, followed by ducks (8%) and a small number of quail, pigeons and geese. Free range ‘backyard’ and scavenging poultry, that are traditionally reared by rural women and children, still play an important role in generating family income, in addition to improving the familys diet with eggs and meat. Productive and reproductive performance of indigenous birds is relatively very low (35–40 eggs and 1–1.5 kg meat per bird per year), but genetic improvements by selective breeding, along with adequate nutrition and proper management, looks promising and quite possible. Commercial poultry production in Bangladesh, is conducted on an industrial scale and is growing tremendously in spite of recent difficulties but is expected to make a significant contribution to the economic development of the country. A national poultry policy is expected to be approved by the government shortly, which, when implemented, will improve the organization of production and marketing, allowing increased stability and security of output throughout the year. In addition, efforts should be taken to ensure safety standards of poultry meat and eggs for human consumption. Experts from the government, research institutes, universities, NGOs and other relevant sectors need to work in a collaborative manner in order to allow sustainable production and fight challenges jointly when they appear from time to time. Corresponding attention to research and development will allow the poultry sector to flourish in Bangladesh. As government funding is limited, industrialists need to come forward either to establish their respective research facilities or to provide funds to universities and research institutes in order to undertake research works of national and international importance.

Localization of apoptotic cells in the cystic ovarian follicles of cows: A DNA-end labeling histochemical study

We examined the frequency of apoptosis in cystic follicular cells to investigate the cause of the delay in regression of cystic follicles. Paraffin sections of healthy antral follicles, early and late atretic ones, and early and late cystic ones were stained using the terminal deoxynucleotidyl transferase (Tdt)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labeling (TUNEL) method to detect apoptotic cells. In the granulosa layer of early cystic and atretic follicles, TUNEL-positive cells were evident. In the theca interna of both early and late atresia, high frequencies of TUNEL-positive cells were observed. In the theca interna, a high frequency of TUNEL-positive cells was noted in the early cystic follicles, whereas their frequency decreased in late cystic follicles. These results suggest that apoptosis occurs in the granulosa and theca interna cells of cystic as well as atretic follicles, but the frequency of apoptosis in theca interna cells decreases in late cystic follicles, which may be responsible for the delay of follicular regression.

Changes in the expression of gallinacins, antimicrobial peptides, in ovarian follicles during follicular growth and in response to lipopolysaccharide in laying hens (Gallus domesticus)

The aim of this study was to identify the types of gallinacin genes (GALs) expressed in ovarian follicles and to determine the changes in their expression during follicular growth and in response to lipopolysaccharide (LPS). Follicles at different stages of growth were collected from laying hens (n = 5) and LPS-injected hens (n = 3). The expression of GALs in the theca and granulosa layers was examined by semi-quantitative RT-PCR. The expression of GAL-1, -2, -7, -8, -10, and -12 in the theca layer and GAL-1, - 8, -10, and -12 in the granulosa layer was identified in white and yellow follicles. The expression of these genes was not changed in the theca and granulosa layers during follicular growth except for a decrease in that of GAL-1 in theca. The expression of GAL-1, -7, and -12 in the theca layer of the third largest follicles was increased in response to LPS at a dose of 1 mg/kg body weight and this increase was induced within 3 h and maintained until 12h postinjection. Granulosa layers did not respond to LPS until 12h injection. These results show that six and four types of GALs are expressed in the theca and granulosa layers of healthy follicles respectively, and their levels do not change with follicular growth except for GAL-1 in theca. Elevated levels of GAL-1, -7, and -12 expression in theca in response to LPS suggest that the theca cells expressing these GALs function to eliminate LPS-containing bacteria.

Effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokines and the subsequent recruitment of immunocompetent cells in the oviduct of laying and molting hens

The goal of this study was to examine whether lipopolysaccharide (LPS) induces the expression of proinflammatory cytokines and chemokines and recruits T cells in the lower part of the oviduct, and whether that response to LPS is different between the laying and molting phase. White Leghorn laying and molting hens were intravenously injected with saline (control) or LPS. The uterus and vagina of oviducts were collected 3 or 6 h after injection, and used for reverse transcription PCR analysis of IL-1β, IL-6, IL-8 (CXCLi2), and lymphotactin (Lptn), and for immunohistochemical analysis for the frequency of CD4+ and CD8+ T cells. The expressions of IL-1β, IL-6, and CXCLi2 in the uterus and that of IL-1β in the vagina were upregulated in response to LPS 3 or 6 h after injection in both laying and molting hens. The CXCLi2 expression in the vagina was upregulated by LPS in laying hens, whereas those effects of LPS were not significant in molting hens. Expression of Lptn showed a tendency to be downregulated after 3 h, with recovery by 6 h after LPS injection. The frequency of CD4+ T cells tended to increase in response to LPS after 6 h in the lamina propria of the uterus and vagina in both laying and molting hens. The CD8+ T cell frequencies in the lamina propria of the uterus and vagina of laying hens increased in response to LPS after 6 h. However, in the molting hens, LPS stimulation resulted in CD8+ T cell increase in the vagina only and not in the uterus. These results suggest that expressions of proinflammatory cytokines and CXCLi2 chemokine are upregulated in association with T cell recruitment in response to LPS in the lower part of the oviduct, although CD8+ T cells in the uterus may be depressed during the molting phase. These immunoresponses may play roles in the defense against infection of the oviduct.

Immunolocalization of avian β-defensins in the hen oviduct and their changes in the uterus during eggshell formation.

The aim of this study was to examine whether avian beta-defensin proteins (avbetaDs) exist in the oviduct, and whether those in the uterus are secreted to the eggshell membrane and eggshell. The oviducts of White Leghorn hens at different times of egg formation, eggshell membrane, and eggshell were used. The presence of immunoreactive (ir) avbetaD-3, -11, and -12 was examined by immunohistochemistry and western blot. Two or three types of avbetaDs were identified in the mucosal surface epithelial cells in each oviductal segment. The density of ir-avbetaD-3 and -12 in the uterus was decreased after the egg entered this segment. Western blot analysis confirmed the presence of ir-avbetaD-3, -11, and -12 in the uterus. In the eggshell membrane, only ir-avbetaD-3 was detected on the surface of fibers at the outer layer of the membrane. The ir-avbetaD-3, -11, and -12 were identified in the eggshell matrix by western blot. These results suggest that the surface epithelial cells are the major sites where avbetaDs proteins exist, and the avbetaDs secreted by the uterus cells are likely to be incorporated in the eggshell membrane and eggshell. These avbetaDs may play roles in the innate host defense of the oviduct and egg surface.

Existence of functional lingual antimicrobial peptide in bovine milk.

The lingual antimicrobial peptide (LAP) belongs to the beta-defensin family in cattle and is localized in epithelial cells of alveoli in mammary glands. The aim was to investigate whether LAP is secreted into milk and whether the secreted LAP has antimicrobial activity. Decaseinated bovine skim milk was applied to sample extraction cartridges, and the eluate was used for competitive enzyme immunoassay and Western blotting to test for the presence of LAP in milk. After tricine-SDS PAGE, the gel was stained using the periodic acid-Schiff reaction to examine the possibility of glycosylation of LAP. The eluate obtained from the sample extraction cartridges was subjected to a LAP antibody-coupled affinity column, after which the antimicrobial activity of its eluate against Escherichia coli was investigated with radial diffusion plate assay and colony-forming unit enumeration following the culture of bacteria with the sample. The immunoreactive LAP was detected in the eluate by competitive enzyme immunoassay (optical density = 0.437 +/- 0.012 vs. 0.468 +/- 0.016). In the Western blotting analysis, immunoreactive bands were seen around 8, 14, and 17 kDa. The bands at 14 and 17 kDa, but not 8 kDa, were periodic acid-Schiff reaction-positive. The eluate of LAP antibody-coupled affinity column had antimicrobial activity against E. coli (cfu/control = 0.17 +/- 0.18). These results suggest that bovine milk contains functional LAP-like substances that exert antimicrobial activity.

Intramammary challenge of lipopolysaccharide stimulates secretion of lingual antimicrobial peptide into milk of dairy cows

Lingual antimicrobial peptide (LAP) belongs to the beta-defensin family in cattle and is found in bovine milk. However, it is unclear whether LAP is involved in the early immune response to mammary infection. The aim of the study was to investigate the changes of LAP concentration in milk after intramammary challenge with lipopolysaccharide (LPS), the gram-negative bacteria cell membrane component, in dairy cows. Milk was collected before and after LPS or phosphate-buffered saline (control) challenge every hour for 12 h on d 0 and twice daily from d 1 to 7. Somatic cell count (SCC), LAP concentration, and lactoperoxidase (LPO) activity in the milk were measured. Somatic cell count started to increase at 2 h postchallenge and remained high until d 5 (694 +/- 187 x 10(3 )to >1,000 +/- 0 x 10(3) cells/mL at d 0; >1,000 +/- 0 x 10(3) cells/mL at d 1 to 3; 684 +/- 194 x 10(3 )to 829 +/- 108 x 10(3 )cells/mL at d 4; 527 +/- 197 x 10(3 )to 656 +/- 145 x 10(3 )cells/mL at d 5). Somatic cell count increased in the control cows, although the levels were lower compared with those in the LPS challenge group. The LAP concentration in milk increased significantly at 2 h post-LPS-challenge and was maintained at high levels until d 2 (8.6 +/- 0.6 to 17.5 +/- 2.3 nM). In the control cow infused with phosphate-buffered saline, there was no increase of LAP concentration in milk (5.1 +/- 0.6 to 7.2 +/- 0.8 nM). Increase of LPO activity in the milk was observed at 6 h after LPS challenge and continued until d 3 (4.7 +/- 0.3 to 9.4 +/- 1.1 U). No increase of LPO activity was observed in the milk of control cows. The increase and subsequent decrease in LAP concentration after LPS challenge occurred earlier than those of LPO activity. In multiparous cows with LPS infusion, there was a significantly negative relationship between the days leading to the basal levels in LAP concentration and LPO activity (r = -0.75). These results suggest that LPS induces secretion of LAP into milk within hours and that LPO may have a synergistic antimicrobial function with LAP in mammary glands of dairy cows.