The efficacy of apical-out chicken enteroids as a model for screening eubiotics
Date
2024
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Antibiotic restrictions in food animal production have led to the need for alternatives to achieve comparable effects. Study of feed additives to replace antibiotics, termed eubiotics, are often studied in vivo. In vitro models however have seen interest due to their lower cost and higher throughput while still mimicking the intestine in certain aspects. A recently developed apical-out chicken enteroids model was evaluated for its ability to serve as a functional representative of the in vivo chicken intestine for purposes of studying tight junction barrier permeability, inflammatory response, and oxidative stress. In addition, five eubiotics of monobutyrin, calcium butyrate, monolaurin, carvacrol, and curcumin were studied through these aspects of the intestine to identify their potential as beneficiaries to the broiler intestine. Enteroids were isolated from Cobb500 broiler chicks at embryonic age 18 with enteroids then transferred to T75 flasks for 24 hours at 37.5°C at 5% CO2. On day 1 of culture enteroids were transferred to well plates and then media was changed along with eubiotic addition on day 2. After 24-hour incubation, enteroids were challenged with either ethylenediaminetetraacetic acid (EDTA), lipopolysaccharides (LPS), or menadione. EDTA and LPS additions were followed by use of fluorescein isothiocyanate-dextran (FD4) to determine epithelial integrity and menadione followed by CellRox to identify reactive oxygen species (ROS) presence. PrestoBlue reagent was also utilized as a measurement of viability. Gene expression was determined through isolation of RNA from treated enteroids with and without eubiotic and/or challenge followed by cDNA preparation and evaluation through RT-PCR. Enteroids were found to exhibit peak viability starting at day 3 of culture, with prior days necessary to achieve optimal development. Expression of selected markers for stem cells, tight junction, and inflammation had significant changes, with stem cell expression decreasing and tight junction and inflammation expression increasing over time. Both LPS and menadione challenges resulted in significant increases in FD4 permeability and ROS generation, respectively. LPS additionally significantly reduced the mRNA expression of tight junction genes in addition to significantly increasing inflammatory cytokine expression, while menadione significantly decreased antioxidant gene expression. All tested eubiotics showed a decrease in enteroid integrity as dosages increased in concentration, with viability determined by PrestoBlue either showing increases or no change. Monobutyrin, calcium butyrate, and monolaurin each significantly decreased epithelial permeability to FD4, with all having beneficial impacts towards tight junction expression in addition to monobutyrin and calcium butyrate towards preventing LPS induced inflammation. Carvacrol and curcumin each reduced ROS generation induced by menadione, as well as increased expression of select antioxidant enzymes. These results together suggest that this apical-out chicken enteroids model sufficiently models tight junction capacity, inflammation, and oxidative stress in an in vitro setting. Additionally, monolaurin effectively protects the epithelial integrity of the enteroids along with monobutyrin and calcium butyrate achieving the same in addition to reducing inflammatory status under challenge. Furthermore, carvacrol and curcumin each alleviate oxidate stress in the face of challenge conditions.
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Keywords
Monobutyrin, Enteroids, EDTA, Eubiotics, Permeability