The crypt villi are the major absorptive and protective units of intestine. Hence, understanding their physiology can provide insight into their role in regulating nutrient uptake, growth, and animal health problems. Taking advantage of the self-repairing ability of the villus crypts, we developed a simple method to generate avian villus enteroids that does not involve many complex/ supportive manipulations such as the use of different growth factors, conditioned media, and support with extracellular matrix gels. Each of the enteroids represent a prototype unit of mucosal villus that contained different cell types and show cell turnover and shedding, typical of villi [27, 28]. The cell renewal, shedding and extrusions have been associated to active migration of cells along the villus which is associated with intestinal homeostasis . Cell shedding by the enteroids was evident by the accumulation of a large number of free cells on subsequent days of culture. The enteroids also showed cell proliferation evident from cell labelling studies which may be responsible for their growth, expansion and budding activities. The presence of different cell types such as the epithelial, goblet, entero-endocrine cells, and the cells that produce antimicrobial peptides, typical of villi [29, 30], were evident using their respective markers. The antibodies against both keratins I and II which are the markers of epithelial cells [31, 32], were strongly reactive with the enteroids as was the antibody against Na-K-ATPase, an ion channel protein, responsible for the maintenance of intestinal health such as the integrity of epithelial tight junction, cell motility and polarization [33, 34]. Decreased levels of Na-K-ATPase activities in the basolateral membrane of intestinal epithelial cells has been linked to chronic intestinal inflammation and malabsorption problems . The enteroids also showed the presence of cadherin, a tight junction associated adhesion protein that maintains epithelial barrier function [26, 36]. The mucin producing goblet cells were identified by their reactivity with SNII lectin [24, 37] and an anti mucin antibody. Of other specialized cells, the identity of enterochromaffin cells were indicated by their reactivity with antibodies specific to serotonin, chicken tryptophan hydroxylase, and chicken chromagranin A which are the markers of these cells . Lysozyme positive cells in the enteroids were indicated by their reactivity to an anti-lysozyme antibody. Paneth cells produce lysozyme but their presence in chickens has been controversial [39, 40] however, in a recent report, Wang et al.  showed the presence of these cells in the intestinal crypts. The location of lysozyme producing cells in the enteroids appeared to be surface associated. Many of the specialized cells such as goblet cells and enteroendocrine cells which occur in low numbers, appeared to be scattered or occur as clusters over the organoids. The enteric organoids were positive for alkaline phosphatase, an enzyme that is associated with enterocytes and implicated in the regulation of fatty acid absorption and protection of intestine against bacterial invasion . Alkaline phosphatase attenuates gut inflammation such as in colitis and is amenable to modulation by nutritional factors [43–45]. However, our preliminary tests with 3 different factors, cGH, DSS, and serotonin, showed no modulation of alkaline phosphatase activity of the enteroids; nonetheless, the measurement of alkaline phosphatase can be a useful marker for enteroid function. Based on these characteristics, the villus enteroid can serve as suitable ex vivo model to study villus functions and its regulation.
For its applications in poultry research such as to understand the effect of nutrients, screening of antibiotic alternatives, and the pathogenesis of enteric diseases, the villus organoids can serve as test models thus, may save both cost and time since they can be generated rapidly within days. Therefore, we investigated the effect of some selective growth factors and chemicals that are likely to affect intestine assessing the gross changes in the morphologies of the enteroids. Our results showed both epidermal growth factor (EGF), a major signaling protein implicated intestine development and repair [46, 47] and insulin like growth factor-1 (IGF-1) that causes intestinal proliferation , both, promoted undulation and budding of the organoids within 24–48 h of treatment although the former was much more effective compared with the later. However, the bone morphogenetic protein-2 (BMP-2), another signaling protein and morphogen  known to play role in intestinal epithelial differentiation  did not produce any significant enterotrophic effect. BMP’s effect on intestine has been suggested to be on the level of mesenchymal cells producing secretory cell differentiation . Thyroxin, similarly, produced no significant morphological effect on the enteroids but dexamethasone produced some shrinkage of epithelial cells without much effect on central cell mass. There are conflicting reports of the effect of thyroid hormones on intestinal epithelial cells with respect to growth although they promote maturation of intestinal mucosa and help in calcium transport process . Previously, using enterocyte culture, we observed the cells treated with thyroxin showed some morphological changes leaning towards more cuboidal shapes compared with the controls ; however, it was not possible to discern any such change from the observation of the whole enteroids. The effect of dexamethasone, a synthetic glucocorticoid, was to some extent consistent with the observations of Urayama et al.  who have reported its shrinking effects on rat small intestinal cells. Serotonin, which is a product of enterochromaffin cells and other neural cells innervating gastrointestinal tract, affects its motility, and modulates Na+/K + exchange system [54–56]. However, serotonin produced little to no morphological changes in the organoids nor did it have any effect on their alkaline phosphatase activities that may be associated to Na-K-ATPase system. The chicken growth hormone (cGH) produced neither any morphological change nor affected alkaline phosphatase activity of the enteroids although growth hormone is known to stimulate growth of intestinal mucosa and the proliferation of intestinal stem cells . Trans-retinoic acid exhibited no morphological effect on the enteroids but the vitamin D3 (calcitriol) appeared to shrink their central mass. Inhibitory and anti-proliferative effect of trans-retinoic acid has been reported in literature , but it was not evident from our results. The effect of vitamin D3 on proliferation or differentiation of enterocytes is little known although it plays a significant role in the absorption of calcium and phosphorous in the intestine . Mycotoxins are fungal metabolites that are toxic to intestine . Compared at the same concentration levels, both aflatoxin B1 and cytochalasin B caused lethal changes resulting in the fragmentation of enteroids within 24 hours of treatment whereas deoxynivalenol (DON) appeared to be relatively less lethal and took longer incubation time, up to 48 h, to produce similar effects. The effects of mycotoxins are presumed to be due to their interfering effects on protein synthesis and cell division, and they disrupt intestinal cell barrier producing cellular apoptosis [61–63] which lead to their degeneration. Similarly, both Staphylococcus aureus and Clostridium perfringens epsilon enterotoxins were lethal to the enteroids and produced severe damage. Neither Salmonella typhimurium lipopolysaccharide nor bacterial peptidoglycan produced any lethal effect in the organoids although both of these products are proinflammatory in many systems . In a previous study with chicken enterocytes, we did not observe any significant effect of LPS on the enterocytes  but Salmonella infection has been reported to affect intestinal organoids causing their morphological changes and disrupting epithelial tight junctions . However, it is not known whether the effect of Salmonella can be due to LPS or to the growth and invasion of the bacteria per se. Similarly, the peptidoglycan was ineffective in damaging the enteroids. The Staphylococcus aureus enterotoxin is well known for its wide range of damaging effects on intestine  and the enterotoxin of Clostridium perfringens has been identified as major virulent factor in the cause of necrotic enteritis in livestock including poultry [67–70] and the Clostridium difficile toxin disrupts the epithelial barrier function of human intestinal organoids . Both of the enterotoxins caused lethal effect on the enteroids.
The effect of some metabolic modulators, indomethacin, a prostaglandin inhibitor, PMA, a protein kinase C activator, and the monensin, an ionophore, anticoccidial antibiotic, on the enteroid morphologies were tested. Indomethacin produces ulcers in human and rat small intestines  however, it appeared only to shrink epithelial aspects of the enteroids whereas PMA showed no significant effect on the enteroids although our previous study with the effect of PMA on enterocytes showed significant cachectic changes in the enterocytes [23, 24]. Monensin, on the other hand, damaged the organoids, however, it has been a preferred anticoccidial drug in the poultry and some early studies suggested its toxic and necrotic effect in the intestine . Tetramethyl thiuram disulfide (thiram) is a fungicide and an endocrine disruptor, produces many toxic effects in chickens including gastrointestinal problems and growth retardation . Thiram exhibited severe toxic effect on the enteroids leading to their disintegration but sodium dextran sulfate, a widely used inducer of experimental colitis, showed no lethal effect on the enteroids although its effect in vivo is attributed to the changes in the mucosal permeability activating inflammatory cells [75–77]. Overall, these results highlight how different modulators may affect the intestinal villi and the villus enteroids may contribute to understand their mechanisms of action.