Tissue-, age- and dose-dependent changes in avian ß-defensin and LEAP2 mRNA abundance in the intestines of Salmonella Typhimurium challenged broilers.

Salmonella is a pathogen normally found in the gastrointestinal tract of poultry. The objective of this study was to determine changes in avian ß-defensin (AvBD) and liver-enriched antimicrobial peptide 2 (LEAP2) mRNA following Salmonella challenge. Day of hatch chicks were challenged with 106, 107 or 108 colony-forming units (cfu) of Salmonella typhimurium. There were dose-, tissue- and age-specific changes in AvBD and LEAP2 mRNA. At 1-day post-infection (dpi) there was a transient upregulation of AvBD1, 8, 10 and 12 mRNA in the 108 cfu group. At 5 dpi, all seven AvBD mRNA were downregulated in the ileum, while only AvBD1, 6, 10 and 11 mRNA were downregulated in the jejunum and AvBD6, 8, 10, 12 and 13 were downregulated in the cecum. At 7 dpi, there was downregulation of all seven AvBD mRNA in the duodenum and downregulation of selected AvBD in the jejunum, ileum and cecum. LEAP2 mRNA was downregulated at all doses of Salmonella in the cecum at 1 dpi and in the ileum at 5 dpi. In summary, Salmonella infection caused an initial upregulation followed by a downregulation of AvBD mRNA.

Supplemental methionine sources have a neutral impact on oxidative status in broiler chickens.

To determine the effect of different dietary Met sources on oxidative status, male Cobb 500 broiler chickens were fed from day of hatch to 26 days of age (d26) a diet deficient in sulfur amino acids (control) or a diet containing 0.22% DL-Met, 0.22% L-Met or 0.31% Met precursor, DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA) to meet the Met + Cys requirements. Liver, breast muscle, duodenum, jejunum, and ileum were collected at day 10 (d10) and d26 to assay markers of oxidative stress, including total glutathione (TGSH), oxidized glutathione (GSSG), reduced glutathione (rGSH), protein carbonyls, thiobarbituric acid reactive substances (TBARS) and ferric reducing/antioxidant power (FRAP). In breast muscle, TGSH and rGSH were greater in L-Met and DL-HMTBA groups than in the control group (p < 0.05). An interaction of treatment and age was observed for TGSH in ileum (p = 0.01) and jejunum (p = 0.01), for GSSG in jejunum (p < 0.001), and for rGSH in ileum (p = 0.02). The ratios of rGSH to GSSG and GSSG to TGSH, which define oxidative status, were not affected by Met source. Protein carbonyls varied among groups in jejunum (p = 0.05) and breast muscle (p < 0.001), but were in the normal physiological range. No difference among treatment groups was observed for TBARS and FRAP in different tissues. Age effects were observed in all tissues for multiple oxidative stress markers. In conclusion, consuming different sources of supplementary Met did not alter the oxidative status in several tissues of broilers. Met + Cys deficiency did not compromise antioxidant capacity of chickens although growth was retarded.

mRNA expression of amino acid transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1 in the intestine and liver of posthatch broiler chicks.

Amino acid (AA) transporter proteins are responsible for the movement of amino acids in and out of cells. Aminopeptidase cleaves AAs from the N-terminus of polypeptides making them available for transport, while PepT1 is a di- and tripeptide transporter. In the intestine, these proteins are present on the brush border and basolateral membranes of enterocytes, and are essential for the uptake of AAs into enterocytes and their release into circulation. The purpose of this study was to determine the level of transcription of these genes after hatch in 3 regions of the small intestine, the ceca, and liver. Heritage broiler chicks (n=5) were sampled at day after hatch and days 3, 5, 7, 10, 12, 14, 17, and 21 posthatch, and mRNA expression level was measured using absolute quantitation. The small intestine (duodenum, jejunum, and ileum) expressed the largest quantities of each gene tested. The expression in the ceca and liver was 1 to 3 orders of magnitude less than that of the small intestine. The expression of basolateral transporters in the small intestine was more constant over days posthatch than the expression of brush border transporters. In the ceca the expression of the brush border transporters decreased over the sampling period, while expression of basolateral genes was relatively constant. In the liver the expression of Na+ independent cationic and zwitterionic amino acid transporter (bo,+AT), Na+ independent cationic amino acid transporter 2 (CAT2), excitatory amino acid transporter 3 (EAAT3), and the heavy chain corresponding to the bo,+) system (rBAT) significantly decreased at 12 days posthatch; however, the expression of Na+ independent cationic and Na+ dependent neutral amino acid transporter 1 (y+LAT1), Na+ coupled neutral amino acid transporter 1; (SNAT1), and Na+ coupled neutral amino acid transporter 2 (SNAT2) significantly increased at day 5 posthatch compared to day 1 and these levels remained throughout the rest of the sampling period. The current results suggest that at 1 day posthatch chicks are capable of AA processing and transport in the intestine as well as the liver. Additionally the ability of the ceca in transporting AA from the lumen may decrease with age. The liver should be capable of amino acid transport, but its capabilities may be more specific since the expression of several transporters in this organ is either absent or very low.

Temporal transcriptome analysis of the chicken embryo yolk sac.

BACKGROUND: The yolk sac (YS) is an extra-embryonic tissue that surrounds the yolk and absorbs, digests and transports nutrients during incubation of the avian embryo as well as during early term mammalian embryonic development. Understanding YS functions and development may enhance the efficient transfer of nutrients and optimize embryo development. To identify temporal large-scale patterns of gene expression and gain insights into processes and mechanisms in the YS, we performed a transcriptome study of the YS of chick embryos on embryonic days (E) E13, E15, E17, E19, and E21 (hatch). RESULTS: 3547 genes exhibited a significantly changed expression across days. Clustering and functional annotation of these genes as well as histological sectioning of the YS revealed that we monitored two cell types: the epithelial cells and the erythropoietic cells of the YS. We observed a significant up-regulation of epithelial genes involved in lipid transport and metabolism between E13 and E19. YS epithelial cells expressed a vast array of lipoprotein receptors and fatty acid transporters. Several lysosomal genes (CTSA, PSAP, NPC2) and apolipoproteins genes (apoA1, A2, B, C3) were among the highest expressed, reflecting the intensive digestion and re-synthesis of lipoproteins in YS epithelial cells. Genes associated with cytoskeletal structure were down-regulated between E17 and E21 supporting histological evidence of a degradation of YS epithelial cells towards hatch. Expression patterns of hemoglobin synthesis genes indicated a high erythropoietic capacity of the YS between E13 and E15, which decreased towards hatch. YS histological sections confirmed these results. We also observed that YS epithelial cells expressed high levels of genes coding for plasma carrier proteins (ALB, AFP, LTF, TTR), normally produced by the liver. CONCLUSIONS: Here we expand current knowledge on developmental, nutritional and molecular processes in the YS. We demonstrate that in the final week of chick embryonic development, the YS plays different roles to support or replace the functions of several organs that have not yet reached their full functional capacity. The YS has a similar functional role as the intestine in digestion and transport of nutrients, the liver in producing plasma carrier proteins and coagulation factors, and the bone marrow in synthesis of blood cells.

Expression of nutrient transporters in duodenum, jejunum, and ileum of Eimeria maxima-infected broiler chickens.

The uptake of amino acids is mediated by active transporters located on the basolateral and brush border membranes of intestinal epithelial cells. The current study investigated the expression of amino acid transporters (AAT) and other genes in the intestine of chicks infected with Eimeria maxima. At 7-day postinfection (PI), tissue from each intestinal segment (duodenum, jejunum, and ileum) was taken from birds inoculated with 3 × 10(3) oocysts/bird and processed to recover RNA. Analysis of gene expression was performed using real-time reverse transcription polymerase chain reaction (qRT-PCR). Results were given as relative expression using ß2-microglobulin as an endogenous control. All the genes studied were expressed in three segments of the intestines, and expression of the genes was altered by infection with E. maxima. Even though the jejunum is considered the parasite's primary predilection site, there was no segment-related difference in expression of most of the genes studied. The antimicrobial peptide (LEAP2) was downregulated in all three segments of the intestine. The results also demonstrate that transporters associated with brush border membranes were downregulated while transporters associated with the basolateral membranes were upregulated and that E. maxima alters the expression of AAT and LEAP2 throughout the small intestine.

The mRNA expression of amino acid transporters, aminopeptidase N, and the di- and tri-peptide transporter PepT1 in the embryo of the domesticated chicken (Gallus gallus) shows developmental regulation.

The mRNA expression profile for 10 amino acid transporters, the di-and tri- peptide transporter (PepT1), and aminopeptidase N (APN) during chick embryogenesis was determined. Fertilized eggs were sampled at d 9, 11, 15, 17, 19, and 20 of incubation. Three to 4 embryos were sampled at each time period. At d 9 and 11, the entire intestine was collected due to its undifferentiated appearance. The ceca, duodenum, midgut, and liver were sampled at d 15, 17, 19, and 20. Gene expression was measured using absolute quantitation quantitative reverse-transcription PCR. In the liver, all genes except for PepT1 were expressed at most time points. At d 9, only the expression of Na⁺-independent cationic amino acid transporter 1, Na⁺-independent cationic amino acid transporter 2, and excitatory amino acid transporter 3 was detectable in the intestine, but by d 11, all genes associated with transporters of the basolateral surface were expressed, and at higher levels than genes associated with brush border transporters. By d 15, all of the genes tested were expressed in the duodenum, midgut, and ceca at high levels that remained relatively constant until d 20. Statistical analysis shows that at d 15, 17, 19, and 20 there is a significant interaction between the 2 main effects (days of incubation and region of the gut); therefore, it is likely that gene expression in different regions of the gut is dependent on the age of the embryo. At d 9 and 11, the gut may not function in amino acid uptake from the lumen and possibly relies on other structures such as the yolk sac. As the gut matures and protein becomes available in the lumen, amino acid transporters become highly expressed in all parts of the intestine. The data suggest that by d 15 of embryo development the gut may be capable of amino acid absorption.

Early intestinal development of chickens divergently selected for high or low 8-wk body weight and a commercial broiler.

The early posthatch period is crucial to intestinal development, shaping long-term growth, metabolism, and health of the chick. The objective of this study was to determine the effect of genetic selection on morphological characteristics and gene expression during early intestinal development. Populations of White Plymouth Rocks have been selected for high weight (HWS) and low weight (LWS) for over 63 generations, and some LWS display symptoms of anorexia. Intestinal structure and function of these populations were compared to a commercial broiler Cobb 500 (Cobb) during the perihatch period. Egg weights, yolk-free embryo BW, yolk weights, and jejunal samples from HWS, LWS, and Cobb were collected on embryonic day (e) 17, e19, day of hatch, day (d) 3, d5, and d7 posthatch for histology and gene expression analysis. The RNAscope in-situ hybridization method was used to localize expression of the stem cell marker, olfactomedin 4 (Olfm4). Villus height (VH), crypt depth (CD), and VH/CD were measured from Olfm4 stained images using ImageJ. mRNA abundance for Olfm4, stem cell marker Lgr5, peptide transporter PepT1, goblet cell marker Muc2, marker of proliferation Ki67, and antimicrobial peptide LEAP2 were examined. Two-factor ANOVA was performed for measurements and Turkey's HSD was used for mean separation when appropriate. Cobb were heaviest and LWS the lightest (P < 0.01). at each timepoint. VH increased in Cobb and CD increased in HWS compared to LWS (P < 0.01). PepT1 mRNA was upregulated in LWS (P < 0.01), and Muc2 mRNA was decreased in both HWS and LWS compared to Cobb (P < 0.01). Selection for high or low 8-wk body weight has caused differences in intestinal gene expression and morphology when compared to a commercial broiler.

Profiling intestinal stem and proliferative cells in the small intestine of broiler chickens via in situ hybridization during the peri-hatch period.

Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5, and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.

Temporal changes of genes associated with intestinal homeostasis in broiler chickens following a single infection with Eimeria acervulina.

Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d postinfection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.

Re-engineering aryl methylcarbamates to confer high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

To identify potential human-safe insecticides against the malaria mosquito we undertook an investigation of the structure-activity relationship of aryl methylcarbamates inhibitors of acetylcholinesterase (AChE). Compounds bearing a ß-branched 2-alkoxy or 2-thioalkyl group were found to possess good selectivity for inhibition of Anopheles gambiae AChE over human AChE; up to 530-fold selectivity was achieved with carbamate 11d. A 3D QSAR model is presented that is reasonably consistent with log inhibition selectivity of 34 carbamates. Toxicity of these compounds to live Anopheles gambiae was demonstrated using both tarsal contact (filter paper) and topical application protocols.

Expression of genes associated with apoptosis in the residual yolk sac during the perihatch period of broiler chicks.

The yolk sac (YS) consists of the yolk and the surrounding YS tissue, which provides essential nutrients and physiological functions for the developing embryo. After the YS is internalized into the abdominal cavity of the embryonic chick, the YS starts to degrade. Apoptosis, or programmed cell-death, is speculated to be the mechanism behind degradation of the YS. The objective of this study was to determine if degradation of the YS tissue was mediated by apoptosis during the perihatch period. The YS tissue was collected from broiler chicks from embryonic d 17 to d 7 posthatch. The mRNA abundance of genes that are involved in the regulation, initiation, and execution of apoptosis were analyzed by qPCR. The mRNA for Bcl2, Bcl2L11, cytochrome C and caspases 3, 6, 7, 8, 9, and 18 all showed a linear response from embryonic d 17 to d 7 posthatch. To confirm the role of apoptosis in the degradation of the YS tissue, a DNA fragmentation assay was performed. Degradation of genomic DNA in the YS tissue started on day of hatch. The characteristic ladder of oligonucleosomal-sized DNA fragments was observed on d 3, 5, and 7 posthatch. The combined gene expression and DNA fragmentation results demonstrate that degradation of the YS posthatch is mediated by apoptosis.

Nutritional stimulation by in-ovo feeding modulates cellular proliferation and differentiation in the small intestinal epithelium of chicks.

Nutritional stimulation of the developing small intestine of chick embryos can be conducted by in-ovo feeding (IOF). We hypothesized that IOF of glutamine and leucine can enhance small intestinal development by promoting proliferation and differentiation of multipotent small intestinal epithelial cells. Broiler embryos (n = 128) were subject to IOF of glutamine (IOF-Gln), leucine (IOF-Leu), NaCl (IOF-NaCl) or no injection (control) at embryonic d 17 (E 17). Multipotent, progenitor and differentiated cells were located and quantified in the small intestinal epithelium between E 17 and d 7 after hatch (D 7) in all treatment groups by immunofluorescence of SRY-box transcription factor 9 (Sox9) and proliferating cell nuclear antigen (PCNA), in-situ hybridization of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) and peptide transporter 1 (PepT1) and histochemical goblet cell staining. The effects of IOF treatments at E 19 (48 h post-IOF), in comparison to control embryos, were as follows: total cell counts increased by 40%, 33% and 19%, and multipotent cell counts increased by 52%, 50% and 38%, in IOF-Gln, IOF-Leu and IOF-NaCl embryos, respectively. Only IOF-Gln embryos exhibited a significance, 36% increase in progenitor cell counts. All IOF treatments shifted Lgr5+ stem cell localizations to villus bottoms. The differentiated, PepT1+ region of the villi was 1.9 and 1.3-fold longer in IOF-Gln and IOF-Leu embryos, respectively, while goblet cell densities decreased by 20% in IOF-Gln embryos. Post-hatch, crypt and villi epithelial cell counts were significantly higher IOF-Gln chicks, compared to control chicks (P < 0.05). We conclude IOF of glutamine stimulates small intestinal maturation and functionality during the peri-hatch period by promoting multipotent cell proliferation and differentiation, resulting in enhanced compartmentalization of multipotent and differentiated cell niches and expansions of the absorptive surface area.

Runting Stunting Syndrome in Broiler Chickens Is Associated with Altered Intestinal Stem Cell Morphology and Gene Expression.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

El síndrome del enanismo infeccioso en pollos de engorde se asocia con alteración de la morfología de las células madre intestinales y la expresión de genes. El síndrome del enanismo infeccioso (con las siglas en inglés RSS) en pollos de engorde se caracteriza por alteraciones en la morfología intestinal y en la expresión de genes, además de retraso en el crecimiento. El objetivo de este estudio fue realizar un estudio retrospectivo de la expresión genética en células madre y células diferenciadas en el intestino delgado de pollitos con el síndrome del enanismo infeccioso. Se analizaron dos modelos diferentes del síndrome del enanismo infeccioso: en pollos de engorde que fueron infectados experimentalmente y en pollos de engorde infectados naturalmente. Los pollitos infectados experimentalmente se expusieron a la cama de parvadas infectadas (RSS-litter chicks), o infectados con astrovirus (RSS-astrovirus chicks). Se adquirieron muestras intestinales de pollitos infectados naturalmente que mostraban signos clínicos del síndrome del enanismo infeccioso de granjas comerciales en Georgia y se llevaron a un laboratorio de diagnóstico avícola (RSS-Clinical-GA) y de granjas en Brasil que tenían antecedentes del síndrome del enanismo infeccioso (RSS-Clinical-BR). Los pollitos de granjas de Brasil (RSS-Clinical-BR) se separaron en aquellos que fueron positivos o negativos para gallivirus de acuerdo con la secuenciación del ADN. Se identificaron la morfología intestinal y el tipo de células intestinales en tejidos archivados fijados con formalina e incluidos en parafina. La hibridación in situ para ARNm específico de células se utilizó para identificar células madre intestinales que expresan olfactomedina 4 (Olfm4), células en proliferación que expresaban Ki67, células absorbentes que expresan el cotransportador 1 de glucosa y sodio (SGLT1) y el transportador de péptidos 1 (PepT1), y células caliciformes que expresan mucina 2 (Muc2). Los pollos expuestos a cama infectada (RSS-litter) y los infectados naturalmente de Georgia (RSS-clinical-GA) mostraron entre un 4% y un 7.5% de criptas quísticas, mientras que los pollos infectados de granjas de Brasil (RSS-clinical-BR) que eran positivos para gallivirus mostraron un 11.7% de criptas quísticas. Los pollos infectados con astrovirus (RSS-astrovirus chicks) y los pollos de Brasil (RSS-clinical-BR) que eran negativos para gallivirus mostraron pocas criptas quísticas. Los pollos expuestos a cama infectada (RSS-litter chicks) y los pollos infectados de Brasil (RSS-clinical-BR) que eran positivos para gallivirus mostraron un aumento en la profundidad de las criptas en comparación con los pollos control o negativos para el gallivirus, respectivamente. No se observó expresión de ARNm de Olfm4 en las células madre de pollitos expuestos a cama infectada (RSS-litter chicks) ni en pollos infectados de Georgia (RSS-clinical-GA), en contraste con la expresión normal de ARNm de Olfm4 en pollitos infectados con astrovirus (RSS-astrovirus chicks) y en pollitos infectados de Brasil (RSS-clinical-BR). Todos los pollos, independientemente del estado de infección, mostraron una expresión normal de ARNm para Ki67 en las células de la cripta, de ARNm para Muc2 en las células caliciformes y ARNm de SGLT1 o PepT1 en los enterocitos. Estos resultados demuestran que el síndrome del enanismo infeccioso, que puede ser inducido por diferentes etiologías, puede mostrar diferencias en la expresión del marcador para células madre Olfm4.

Expression profiles of somatotropic axis genes in lines of chickens divergently selected for 56-day body weight.

The objective of this study was to evaluate mRNA expression of somatotropic axis genes in chickens divergently selected for high (HWS) or low (LWS) body weight at 56 days of age. Gene expression was measured on days 16, 18, and 20 of incubation, day of hatch, and days 3, 7, 28, and 56 posthatch. Pituitary growth hormone mRNA raised from prehatch to posthatch, with a similar profile in both lines. Liver growth hormone receptor (GHR) mRNA was high during embryogenesis, declined to low levels at day 3 posthatch, and then increased to day 56. Expression of liver insulin-like growth factor 1 (IGF-1) mRNA increased sharply by day 28 in line HWS and day 56 in line LWS. Pectoralis major muscle GHR mRNA was greater in line LWS than HWS. Muscle IGF-1 mRNA declined during embryogenesis, increased posthatch, and declined after day 7. IGF-1 mRNA was 1,000-fold greater in embryonic muscle than embryonic liver. Muscle IGF-1 receptor mRNA was greater in line LWS than HWS posthatch. These results demonstrate that genetic selection for high or low body weight has altered the expression profiles of somatotropic axis genes in a line-, age-, and tissue-specific manner.

Proteomic evaluation of chicken brush-border membrane during the early posthatch period.

The chicken small intestine undergoes structural and functional changes during the early posthatch period to accommodate the transition from a lipid-rich diet inside the egg to a carbohydrate- and protein-based diet. Many of the enterocyte brush-border membrane-associated proteins responsible for mediating changes in nutrient utilization are unknown. The objective of this study was to conduct a proteomic analysis of chicken small intestine during the early posthatch period. We isolated brush-border membrane at day of hatch and days 1, 3, 7, and 14 posthatch from the small intestine of 2 genetic lines of broilers that differ in growth performance, and performed 2D gel-electrophoresis. A total of 1693 spots were analyzed by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). In total, 132 different proteins were identified and grouped according to biological function. Of these, there were 10 nutrient transporters, 9 digestive enzymes, and 17 proteins associated with cytoskeletal structure and microvilli organization. The remaining proteins were classified as basolateral membrane (3), endosomal/membrane trafficking (8), signaling (14), metabolic (33), degradative (5), stress-related (5), protein synthesis machinery/mitochondria/nucleus (19), immunologic (1), or unknown (8). Of the spots in which proteins were identified, there were 10 that showed an effect of broiler genetic line on protein spot density (P<0.001) and 19 spots showing a correlation of broiler genetic line x age (P<0.001). Identification of brush-border membrane-associated proteins is an important step in furthering our understanding of digestion and absorption in the chicken.

Expression of avian ß-defensin mRNA in the chicken yolk sac.

The chicken yolk sac (YS) plays an important role in nutrient absorption and immune function for the developing embryo. The avian ß-defensins (AvBD) are cationic peptides that are important members of the innate immune system. The objective of this study was to profile AvBD mRNA expression patterns and distribution of cells expressing AvBD mRNA in the chicken YS. Expression of AvBD1, 2, 7, and 10 mRNA was low at embryonic day 7 (e7), increased to e9 through e13 and then declined to e19. Using in situ hybridization, AvBD10 mRNA was found to be expressed in endodermal epithelial cells, while AvBD1, 2, and 7 mRNA were expressed in heterophils. The developmental expression pattern and distribution of AvBD mRNA in the YS reveals the importance of these genes to protection of the developing chick embryo.

Dietary protein quality and feed restriction influence abundance of nutrient transporter mRNA in the small intestine of broiler chicks.

The objective of this study was to evaluate the effect of dietary protein quality on intestinal peptide transporter (PepT1), amino acid transporter [Na+-independent cationic and zwitterionic amino acid transporter (b(o,+)AT), excitatory amino acid transporter 3 (EAAT3), Na+-independent cationic and Na+-dependent neutral amino acid transporter (y+ LAT2), and Na+-independent cationic amino acid transporter 2 (CAT2)], glucose transporter [Na+-dependent glucose and galactose transporter 1 (SGLT1) and Na+-independent glucose, galactose, and fructose transporter 2 (GLUT2)], and digestive enzyme [aminopeptidase N (APN)] mRNA abundance in 2 lines of broilers (A and B). At day of hatch (doh), chicks from both lines were randomly assigned to corn-based diets containing 24% crude protein with either soybean meal (SBM) or corn gluten meal (CGM) as the supplemental protein source. Chicks were given unlimited access to feed and water. Groups of chicks from both lines were also assigned to the SBM diet at a quantity restricted to that consumed by the CGM group (SBM-RT). Intestinal transporter and enzyme mRNA abundance was assayed by real-time PCR using the absolute quantification method. Abundance of PepT1, EAAT3, and GLUT2 mRNA was greater in Line B (P < 0.03), whereas APN and SGLT1 were greater in Line A (P < 0.04). When feed intake was equal (CGM vs. restricted SBM), a greater abundance of PepT1 and b(o,+)AT mRNA was associated with the higher quality SBM (P < 0.04), whereas a greater abundance of EAAT3 and GLUT2 mRNA was associated with the lower quality CGM (P < 0.01). When feed intake was restricted (SBM vs. SBM-RT), a greater abundance of PepT1 mRNA was associated with the restricted intake (P < 0.04). These data demonstrate that both dietary protein quality and feed restriction influence expression of nutrient transporter mRNA in the small intestine of broiler chicks.

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae.

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.

Functional characterization of the chicken peptide transporter 1 (pept1, slc15a1) gene.

Dietary amino acids can be transported into intestinal epithelial cells as di- and tripeptides by the action of the peptide transporter, PepT1 (SLC15A1). Expression of the chicken PepT1 (cPepT1) gene changes in response to dietary crude protein level; however, the molecular mechanism governing this regulation is unknown. This study analyzed the promoter region of the cPepT1 gene. Using deletion analysis, positive-acting (-314 to -261, -169 to -155, and -120 to -60) and negative-acting (-419 to -386 and -214 to -169) regions were mapped in transfected chick embryo fibroblasts (CEF). The addition of neither amino acids Phe, Arg, or Val, nor the dipeptides Gly-Sar (glycyl-sarcosine), Gly-Pro, Gly-Phe, Met-Pro, Met-Lys or Lys-Lys, had an effect on cPepT1 promoter activity in transfected CEF. The cPepT1 promoter was more active in CEF and primary chicken intestinal cells than in chicken liver cells. This study represents a functional characterization of the molecular regulation of the chicken PepT1 gene.

Gene expression and activity of methionine converting enzymes in broiler chickens fed methionine isomers or precursors.

Common dietary supplemental methionine (Met) sources include DL-methionine (DL-Met) and the Met precursor DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA). For bio-utilization, D-Met and DL-HMTBA are converted into L-Met through oxidation and transamination. The objective of this study was to determine the effect of different dietary supplemental Met sources on gene expression and enzyme activity of Met oxidases in male broiler chickens. Liver, muscle, duodenum, jejunum, and ileum were collected at days 10 (d 10), 21 (d 21), and 26 (d 26) post-hatch from male broiler chickens that were fed a basal diet deficient in sulfur amino acids (SAA) (control), or the control diet supplemented with DL-Met, L-Met, or DL-HMTBA to meet SAA requirements. The mRNA abundance of D-Met oxidase, L-HMTBA oxidase, and D-HMTBA oxidase was measured by real-time PCR, and oxidase activities were measured using colorimetric assays (n = 5). Liver expressed more D- and L-HMTBA oxidase mRNA, while breast muscle and liver expressed more D-Met oxidase mRNA than other tissues. In the liver, DL-HMTBA and L-Met supplementation were associated with greater mRNA abundance of L-HMTBA oxidase compared to the control diet-fed group at d 10 but not d 21 or d 26. DL-HMTBA supplementation, however, was not associated with changes in the mRNA abundance of D-HMTBA oxidase. The Met-deficient diet at d 26 was associated with greater hepatic abundance of DAO mRNA, which is responsible for oxidation of amino acids. Oxidase activities were similar among the Met deficient and Met-supplemented groups. In conclusion, dietary Met supplementation influenced the transcriptional regulation and activity of Met oxidases in a tissue and age-specific manner. Met oxidases may thus act as a determining factor in the bioefficacy of different dietary supplemental Met sources.