The 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 that uses standard cell culture medium that does not involve many special growth factors, conditioned media, and extracellular matrix gel supports to generate avian villus enteroids, that are often used to favor the clonal expansion of the crypt based progenitor cells. Each enteroid appeared to be a prototype unit of mucosal villus containing different cell types and showed cell turnover and shedding typical of the 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 was evident by the accumulation of a large number of free cells around the enteroids on subsequent days of culture. The enteroids also showed cell proliferation, evident from cell labelling studies and were indicative of their growth and budding activities although these activities tend to slow down. The presence of different cell types such as the epithelial, goblet, entero-endocrine cells, and the cells that produce antimicrobial peptides that are typical of villi [29, 30], and were evident from their respective markers. The antibodies against both keratins I and II, which bind to the 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 and 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 presence of enterochromaffin cells were indicated by the reactivity of the 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 as scattered patches of cells. The enteroids 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 also attenuates gut inflammation such as in colitis and is amenable to modulation by nutritional factors [43-45]. However, our results with 3 different factors, cGH, DSS, and serotonin, showed no significant modulation of alkaline phosphatase activity by any of these factors; nonetheless, the measurement of alkaline phosphatase can be a useful marker for enteroid function.
For its applications in poultry research, such as to understand the effect of nutrients, interactions with pathogens, or the screening of antibiotic alternatives, the villus organoids can be generated inexpensively and rapidly within days and serve as test models either as individual or collective units employing their morphological and biochemical changes. To test it, we investigated the effect of selective growth factors and chemicals that are known to affect intestine by assessing the gross changes in the morphologies of the enteroids. Our results showed both epidermal growth factor (EGF) and the insulin like growth factor-1 (IGF-1) to have trophic effect on the organoids. EGF is a major signaling protein implicated in intestine development and repair [46, 47] and insulin like growth factor-1 (IGF-1) causes intestinal proliferation , both of which 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  that has been shown to affect 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 . Since, the enteroids have limited mesenchymal cells, the effect of BMP may not be morphologically discernible. Thyroxin, similarly, produced no significant morphological effect on the enteroids whereas dexamethasone produced some shrinking effect on 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 thyroxin has been shown to 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 apparent from the observation of the whole enteroids. The effect of dexamethasone, a synthetic glucocorticoid, to some extent, was consistent with the observations of Urayama et al.  who reported its shrinking effects on rat small intestinal cells. Serotonin, which is a product of enterochromaffin cells and other neural cells innervating gastrointestinal tract, are known to affect its motility, and possibly modulate Na+/K+ exchange system [54-56]. In this assay, serotonin produced little to no morphological changes in the organoids nor did it have any effect on their alkaline phosphatase activities that could 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 GH is known to stimulate the growth of intestinal mucosa and proliferation of intestinal stem cells . Trans-retinoic acid similarly, exhibited no morphological effect on the enteroids but the vitamin D3 (calcitriol) appeared to shrink the central mass of cells. 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 likely 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 fungal peptidoglycan produced any damaging effect on the organoids although both of these products are proinflammatory in many systems . In a previous study with chicken enterocytes, LPS also did not produce any significant effect on the enterocytes , although, Salmonella infection has been reported to affect intestinal organoids causing their morphological changes and disrupting epithelial tight junctions . It is not known whether the effects of Salmonella were due to their LPS content or due to the growth and invasion of the bacteria per se. 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 a major virulent factor that causes necrotic enteritis in livestock including poultry [67-70]. Besides, the Clostridium difficile toxin disrupts the epithelial barrier function of human intestinal organoids . Both of the enterotoxins produced lethal effect on the enteroids.
With regards to metabolic modulators, the effect of indomethacin, a prostaglandin inhibitor, PMA, a protein kinase C activator, and monensin, an ionophore and anticoccidial antibiotic, on the enteroid morphologies were tested. Indomethacin produces ulcers in human and rat small intestines  although it may be a long term effect of the drug. In the current study, indomethacin appeared to only affect the outer epithelial cells of the enteroids without causing any lethality. Similarly, in a previous study using PMA where we observed significant cachectic effect of it on the enterocytes at concentrations of 1µg/ml or less [23, 24], showed no significant effect on the enteroids. Monensin, an anticoccidial drug used in poultry production caused significant damage to the organoids and in some early studies it was reported to have toxic and necrotic effect on 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 dextran sodium sulfate (DSS), a widely used inducer of experimental colitis, showed neither any damaging effect on the enteroids nor caused any change in their alkaline phosphatase activities. However, the in vivo effect of DSS is attributed to the changes in the mucosal permeability and the activation of inflammatory cells [75-77].