Studies on chickens have suggested that several factors, such as an altered diet or poor husbandry, may influence the intestinal mechanical barrier by altering the depth of the crypts and the height of the villi ^[1][2][225,226]. Light may also be an important environmental factor influencing behaviour, growth, and health in birds.

The height of the villi, the crypt depth, and the ratio between them may also represent the gut mechanical barrier ^[1][2][3][4][215,225,226,227]. Morphometric measurements of villus length and intestinal crypt depth are widely used as factors contributing to the preservation of intestinal homeostasis ^[5][228]. Shorter villus length and larger crypts may lead to a poorer nutrient absorption and consequently harm animal health ^[3][6][7][215,229,230].

It needs to be considered that digestion occurs in birds in the upper part of the small intestine, that is, in the duodenum, and released nutrients are mainly absorbed in the lower part of the small intestine. As a consequence, most nutrient absorption occurs in the jejunum and ileum ^[8][231], which could be affected when both values, the length of the villi and the depth of the crypts, are reduced.

A previous study by Xie et al. ^[3][215] reported that green light and blue light promote better small intestinal mucosal structure, and they revealed that green light resulted in an increase in intestinal villus height and villus-to-crypt ratio and a decrease in crypt depth at an early growth stage (7–21 days) and blue light at a later growth stage (49 days) (Photo no. 3). Similar results have been reported in Arbor Acres broilers ^[9][232], Ross 308 broilers ^[10][233], and Leghorn chicks ^[11][234].

GALT is a key immune system in birds, estimated to comprise more immune cells than any other tissue ^[12][235], with associated structures forming a site that promotes the co-localisation of the many types of immune cells required to initiate and mediate immune function. Immune-associated cells in the intestinal mucosa typically contain intraepithelial lymphocytes (iIELs) and IgA⁺ cells, which are the main immune factors ^[3][215].

Prior studies have suggested that iIEL is a population of T lymphocytes and may be of particular importance as an immunological barrier during mucosal immune response ^[3][13][14][215,236,237].

iIELs comprise a heterogeneous population of cells that are located in the basal and apical parts of the epithelium ^[15][16][238,239]. At hatching, a few IELs are detected and their number increases greatly with age; however, in older birds, their number decreases significantly ^[16][239].

IgA⁺ cells are the main element of humoral immunity in the gastrointestinal tract ^[17][18][240,241] and are often spread from the crypts to the tips of the villi ^[3][215].

It has been shown that sIgA, produced by IgA⁺ cells, is involved in immunological barriers in the gut ^[19][242], contributing to the maintenance of intestinal homeostasis ^[20][243]. Therefore, the number of sIgA and IgA⁺ cells in the small intestine can be considered indicators for the appreciation of intestinal mucosal immunity ^[9][11][21][232,234,244].

In an earlier study, Xie et al. ^[3][215] reported that green light and blue light improved gut mucosal immunity in broiler chickens, and they revealed that the population of iIEL and IgA⁺ cells became evident in the group subjected to green light treatment at an early growth stage (7–21 days) and blue light treatment at a later growth stage (49 days) ^[3][215] (Figure 1).

These results agree with those obtained by Li et al. ^[22][78], namely that green light had a positive influence on IgA⁺ cell counts at 14 days of age in the cecal tonsil of chickens. However, the growth induced by light treatment disappeared after pinealectomy, a finding that suggests that melatonin secreted by the pineal gland under the influence of green light is essential for the improvement of immune function ^[22][78].

Melatonin is capable of indirectly enhancing antioxidant activity and removing oxygen free radicals, which in turn regulate the antioxidant status of organs. Physiological doses of melatonin, for example, have been shown to increase gene expression and enzyme activities of GSH-Px, GSHRd, SOD, and CAT ^[23][168]. Furthermore, melatonin may also decrease iNOS activity in mice ^[24][245].

Melatonin is secreted as a function of photoperiod. Various studies have been conducted, focusing on the influence of monochromatic light on antioxidant activity in cecal tonsils. Li et al. ^[22][78] demonstrated that monochromatic green light promoted GSH-Px, SOD, and T-AOC activities and inhibited the iNOS-positive cell population and MDA content in 14-day-old chicks. However, the enhanced effect of green light disappeared after pinealectomy, indicating a close relationship between the pineal gland, melatonin, and different monochromatic light ^[22][78] (Figure 1).

The chemical barrier of the intestinal mucosa may also be represented by the population of goblet cells, which are single glandular cells distributed along the columnar epithelia ^[25][246]. Their role is to secrete high molecular weight glycoproteins known as mucins ^[26][247]. Mucus, which is secreted by goblet cells, is an integral structural component of the intestine that acts as a protective environment and participates in maintaining local intestinal immune homeostasis ^[27][248], lubrication, and transport between luminal contents and the epithelial lining ^[28][29][30][249,250,251].

So far, 21 different mucin genes have been identified. Of these, Muc2, an essential component for barrier integrity, is highly expressed by intestinal goblet cells ^[31][32][252,253] and plays an important protective role in the gut by preventing foreign bacteria and other pathogens from crossing the intestinal mucosa to come into contact with the cells underlying the epithelial cells ^[33][254]. Muc2⁻/⁻mice have bacteria in direct contact with epithelial cells and spontaneously develop colitis and cancer ^[25][34][35][246,255,256].

Uni et al. ^[36][257] reported that the first goblet cells develop in the small intestine of a broiler chick starting 3 days before hatching (at 18 embryonic days) and contain only acidic mucins at that time. In comparison, Yu et al. ^[25][246] reported that goblet cells in the small intestine first appear at 15 embryonic days, which is approximately 3 days earlier than the time reported by Uni et al. ^[36][257]. This discrepancy in the timing of goblet cell development is most likely attributed to differences in materials and methods used in the studies ^[25][246].

Previous studies have shown that the number of intestinal goblet cells is significantly increased when broilers are reared under monochromatic green light at an early age and under monochromatic blue light at an older age ^[3][215]. This research is similar to that of Yu et al. ^[25][246], in which they proved that monochromatic green light can alter the proportion of goblet cell types and promote the proliferation and maturation of goblet cells in broilers during late embryonic stages. This pattern is similar to that found in other previous studies ^[25][36][246,257] (Figure 1).

The intestinal epithelium serves as a vital natural barrier, safeguarding against the entry of pathogenic bacteria and toxic substances present in the intestinal lumen. However, various stressors such as pathogens, chemicals, and other factors can disrupt the balance of the normal microflora or impair the integrity of the intestinal epithelium. Consequently, the permeability of this natural barrier can be compromised, thereby facilitating the invasion of pathogens and harmful substances. This disruption in the intestinal barrier function can have diverse effects, including alterations in metabolism, impaired nutrient digestion and absorption, and the initiation of chronic inflammatory processes within the intestinal mucosa ^[37][38][258,259].

Stress is an important factor that makes birds vulnerable to potentially pathogenic microorganisms such as E. coli, Salmonella spp., Clostridium spp., Campylobacter spp., etc. This pathogenic microflora in the small intestine competes with the host for nutrients and also reduces the digestion of fats and fat-soluble vitamins due to bile acid deconjugation effects ^[39][260].

A change in the gut microbiota of chickens may influence their immunity and health. However, changes in the gut microbiota of chickens can be influenced by several factors. These factors include maintenance conditions, feed composition, and the presence of antibiotics in animal feed ^[40][261].

Recent studies have been conducted to better understand the effects of different combinations of monochromatic light on changes in the microbial population in the broiler cecum ^[41][262].

Zhang et al. ^[42][205] observed that chickens subjected to monochromatic white light treatment had the highest population of Lactobacillus spp. In a previous study, it was predicted that Lactobacillus spp. could efficiently ferment carbohydrates and inhibit the colonisation of other bacteria by lowering pH levels in the digestive tract ^[42][43][205,263]. Lactic acid bacteria are valued as beneficial bacteria by producing acids (lactic acid) and bacteriocin-like substances ^[41][44][262,264].

Short-chain fatty acids (SCFAs) are the main metabolites produced by the microbiota in the large intestine through the anaerobic fermentation of indigestible polysaccharides, such as dietary fibre and resistant starch ^[45][265]. Butyricicococcus spp., Ruminiclostridium spp., and Ruminococcaceae ^[46][47][48][266,267,268] are SCFA-producing bacteria, and their populations were enriched in chickens subjected to a monochromatic colour combination of green and blue light, which could be responsible for the high level of SCFA in cecal tonsils in chickens ^[42][49][123,205].

Butyricicococcus spp. can produce a significant amount of butyrate, which has important immunomodulatory functions and acts as a modulator of chemotaxis and immune cell adhesion ^[50][269]. Butyrate, as a modulator of gut mucosal immunity, can regulate IL6 ^[51][270] or TNF ^[52][271] secretion, activate B lymphocytes to promote antibody production, and induce T lymphocyte proliferation ^[43][263]. In addition, a larger population of the genus Faecalibacterium has been characterised as an anti-inflammatory mediator ^[42][53][205,272].

The most negative consequences were found in the case of red monochromatic light treatment, as it promoted the growth of pathogenic bacteria such as Escherichia–Shigella ^[42][205]. Recently, studies have found that Escherichia–Shigella has been recognised as negatively correlated with fat growth and digestibility ^[54][273] and has become a significant cause of morbidity and mortality in broilers ^[42][55][205,274].

The consideration of melatonin secretion by enterochromaffin cells (ECs) in the digestive system is important. Significant progress has been made in understanding the localisation and physiological functions of melatonin in the gastrointestinal tract (GIT) since its discovery ^[56][275]. Numerous reviews have explored various aspects of melatonin in the GI tract ^{[57][58][59][60][61]}[276,277,278,279,280].

Bubenik et al. ^[62][281] utilised specific melatonin antibodies to localise melatonin throughout the entire GIT of rats. Their study demonstrated that melatonin distribution in the GIT corresponds to the presence of serotonin-producing EC cells ^[62][63][64][281,282,283], supporting the hypothesis that melatonin is produced from serotonin in the GI mucosa. Subsequent studies employing radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) confirmed the presence of melatonin in the GIT ^[65][284].

Several authors, particularly in birds, reported a significant diurnal variation in melatonin concentrations in certain GI tissues, with higher plasma levels during the night ^[65][66][67][284,285,286]. Pinealectomy in pigeons reduced midnight melatonin levels by approximately 50% but did not affect midday levels ^[65][284]. Conversely, in rats, pinealectomy lowered melatonin levels in serum but had no impact on GI tissue levels ^[68][287]. While pinealectomy attenuated nighttime melatonin levels in blood, it had no effect on daytime levels, which are likely produced in the GIT ^{[69][70][71][72][73]}[95,209,288,289,290].

The injection or oral administration of exogenous melatonin and its precursor tryptophan led to a significant accumulation of melatonin in the GIT ^{[66][71][74][75]}[285,288,291,292]. The GIT significantly contributes to circulating plasma levels of melatonin, particularly during the daytime ^{[66][76][77][78][79]}[285,293,294,295,296].

The rapid increase in melatonin in the blood following re-feeding ^[80][297] may potentially trigger a speculative pulse that influences the shift in biological rhythms. Changes in locomotion, temperature, and cortisol rhythm have been observed after food intake. Notably, these rhythm shifts were not mediated by the pineal gland, as they persisted even after lesions in the suprachiasmatic nucleus, which regulates the circadian rhythm of pineal melatonin synthesis ^[81][298].

Conversely, pinealectomy or sham pinealectomy had no effect on the reported changes in sleep patterns, suggesting an independent effect unrelated to the pineal gland ^[82][299]. These circadian activity changes related to food intake could be attributed to a pulse of melatonin released from the GIT, which initially enters the peripheral circulation and subsequently affects the circadian clock ^[78][80][295,297].

The considerable variations in melatonin concentrations in GIT tissues appear to be influenced by food intake rather than photoperiodicity, unlike pineal-produced melatonin ^[76][80][83][293,297,300]. Species differences may reflect diurnal and nocturnal tendencies as well as monophasic or polyphasic feeding behaviours. Additionally, variations in digestion occur between monogastric and polygastric animals ^[61][84][85][280,301,302].

Immunohistology and RIA have predominantly localised melatonin in the mucosa of the GIT ^{[56][62][67][79][84][86]}[275,281,286,296,301,303], including the intestinal villi ^[67][86][286,303]. Conversely, lower concentrations and bindings of melatonin have been detected in the submucosa and muscularis ^{[58][67][83][86]}[277,286,300,303]. These findings support the speculation that melatonin is produced by enterochromaffin cells in the mucosa but can act as a paracrine hormone in other segments of the GIT ^[58][277].

The effects of melatonin synthesised in the gastrointestinal tract primarily occur through paracrine mechanisms, while pineal gland-derived melatonin exerts changes through humoral and neurocrine pathways. In addition to its biorhythmic, antioxidant, and immunomodulatory effects, melatonin influences motor functions, microcirculation, and mucosal cell proliferation in the gastrointestinal tract.

Melatonin secreted in the digestive tract is not regulated by the pineal gland but contributes to overall serum melatonin levels. Together with melatonin from the pineal gland, they synergistically affect the immune system through the aforementioned mechanisms.