Browsing by Author "Cloft, Sara E."

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    Changes with age in density of goblet cells in the small intestine of broiler chicks
    Reynolds, K. L.; Cloft, Sara E.; Wong, Eric A. (Elsevier, 2020-05-01)
    Goblet cells secrete mucin 2 (Muc2), which is a major component of the mucus that lines the intestinal tract and creates a protective barrier between pathogens and the intestinal epithelial cells and thus are important for chick health. The objectives of this study were to determine the age-specific and intestinal segment–specific expression of Muc2 mRNA and changes in the number of goblet cells from late embryogenesis to early after hatch. Small intestinal samples from the duodenum, jejunum, and ileum were collected from Cobb 500 broilers at embryonic day 19 (e19), day of hatch (doh), and day 2 and 4 after hatch. Cells expressing Muc2 mRNA and mucin glycoprotein were detected by in situ hybridization or alcian blue and periodic acid–Schiff staining, respectively. Along the villi, there were many more cells expressing Muc2 mRNA than those stained for mucin glycoprotein. In the crypt, cells expressing Muc2 mRNA did not stain for mucin glycoprotein. There was an increase in the density of goblet cells in the villi and Muc2 mRNA expressing cells in the crypts of the jejunum and ileum from e19 to doh and day 2 to day 4, with no change between doh and day 2. In contrast, in the duodenum, the density of goblet cells in the villi and Muc2 mRNA expressing cells in the crypts remained constant from e19 to day 4. At day 4, the villi in the ileum had a greater density of goblet cells than the duodenum. In the crypt, the ileum had a greater density of Muc2 mRNA expressing cells than the duodenum at doh, and the ileum and jejunum both had greater densities of Muc2 mRNA expressing cells than the duodenum at day 4. These results indicate that the population of goblet cells has reached a steady state by doh in the duodenum, whereas in the jejunum and ileum, a steady-state population was not reached until after hatch.
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    Expression of genes associated with apoptosis in the residual yolk sac during the peri-hatch period of broiler chicks
    Reno, Kaitlyn E.; Cloft, Sara E.; Wong, Eric A. (Elsevier, 2022-08-01)
    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.
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    Outlining a balance-point model of homeostasis in the small intestine of broiler chickens
    Cloft, Sara E. (Virginia Tech, 2022-07-01)
    Since the removal of in feed antibiotics in the past few years commercial poultry production is especially sensitive to the health of the small intestine. Healthy small intestines balance nutrient absorption and defensive barrier functions to ensure the chicken is able to meet the whole-body nutritional needs and is able to help prevent internalization of pathogens or potentially toxic components. This balance can only be maintained under stable conditions. When a disturbance event occurs the intestine imbalances until a new, and less efficient, balance can be achieved. The objective of this dissertation is to propose a novel model to understanding intestinal homeostasis in the face of various disturbance events. Chapter 2 investigated the effects of Runting Stunting Syndrome on broiler chickens in four different groups of chicks displaying clinical symptoms. The major finding in this study was that in two of the four groups the expression of stem cell gene Olfactomedin 4 was absent from the crypt though other functional genes were found to still be expressed there. Chapter 3 characterized intestinal gene expression following a single challenge of Eimeria acervulina in broiler chickens. During Eimeria infection gene expression of multiple host defense peptide genes were decreased compared to uninfected chickens. Further, Eimeria infected chickens increased cell proliferation within the crypt and post-peak infection showed signs of intestinal recovery. Additionally, chapter 3 developed a novel method for visualizing Eimeria as it infects the intestine. In chapters 4 and 5 cell type population changes during the peri-hatch intestinal maturation process were evaluated. Peri-hatch intestinal maturation is critical for the successful transition from embryonic to post-hatch life. Chapter 4 profiled changes in proliferative cells and gene expression of various stem cell marker genes during the peri-hatch period: the last three days of embryogenesis and the first week post-hatch. The stem cell marker gene Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) decreased during the post-hatch period while Olfactomedin 4 increased post-hatch. Both stem cell genes were expressed within the intestinal crypt, though prior to hatch Lgr5 was expressed in the lamina propria and villi as well. Additionally, the marker of proliferation Ki67 gene was expressed in cells throughout the intestine prior to hatch but became restricted to the crypts and along the center of the villi. Chapter 5 assessed the effect of providing probiotics to late term embryos via in ovo feeding (IOF). The effects of IOF were primarily observed on embryonic day 20 (e20), roughly 48 hours after IOF. On e20 the embryos in ovo fed probiotics in saline had increased expression in the ileum of Peptide Transporter 1 (PepT1) a marker gene for enterocytes and Mucin-2 (Muc2) a marker gene for goblet cells compared to non-injected control embryos. Also, on e20 the embryos in ovo fed saline only had numerically increased PepT1 and Muc2 compared to non-injected control embryos. The difference in responses between the probiotic and saline fed embryos on e20 suggests different routes of stimulation. These investigations illustrate various possible scenarios and means of investigating intestinal homeostasis during disturbance events.
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    Profiling intestinal stem and proliferative cells in the small intestine of broiler chickens via in situ hybridization during the peri-hatch period
    Cloft, Sara E.; Uni, Zehava; Wong, Eric A. (Elsevier, 2023-04)
    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.
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    Research Note: Intestinal morphology and gene expression changes in broilers supplemented with lysolecithin
    Cloft, Sara E.; Jia, Meiting; Wong, Eric A. (Elsevier, 2021-07-01)
    Lysolecithin is used as a feed additive to aid fat digestion and absorption in broiler chickens. Previous research has shown that dietary fat source influences how broilers respond to lysolecithin supplementation. Therefore, the objective of this study was to investigate the effect of lysolecithin on a diet formulated with soybean oil on jejunum morphology and expression of selected genes in broiler chickens. Male Cobb 500 chickens were fed a Control diet or the Control diet supplemented with lysolecithin (TRT) from day of hatch to day 28. Jejunal samples were collected at day 10 for morphological and gene expression analysis. Feeding the TRT diet did not affect BW, villus height (VH), crypt depth (CD) or VH/CD ratio compared to Control fed chickens. Differential gene expression in the jejunum was analyzed using a custom microarray. Using a t test, 36 genes were found to be upregulated in TRT fed chickens compared to chickens fed the Control diet. The two most upregulated genes were carbonic anhydrase VII and interleukin 8-like 2, which are associated with healthy intestines. In summary, lysolecithin supplementation in a diet formulated with soybean oil caused no morphological changes but upregulated a number of genes in the jejunum.