The inspected species of birds represent a normal type of cornea, which is composed of five morphological layers; highly squamous epithelium, foremost and anterior basement film bowman's film, lamellated stroma with mucopolysaccharides ground lattice, posterior basement membrane Descemet’s membrane, and lastly a single row of endothelium. Comparable architecture was seen in numerous fowls and in different vertebrates [24, 25].
Concerning avian corneal epithelium of the studied species, it is arranged as a stratified squamous non-keratinized epithelium, a basal columnar cell, followed by polyhedral layers, ranging from two to three, and ending with one layer of flat squamous cells. It is noticed that the characteristic feature of the stratified epithelium declined in the examined epithelial layer from E. albus (forested habitat) to B. Buteo (higher temperature desert area). One explanation for this decrease in the stratification of epithelium layer is accommodation with various habitats, which in turn stimulate the structural modifications in the epithelial layers [26].
As for the epithelium of forested species ibis (Eudocium albus), it acts as excellent protective cover for the rest optical layers due to its highly regenerative capacity during regenerations phases [27]. Similar explanations were reported in diurnal birds such as Circaetus gallicus [28] and Bubo bubo [29]. Furthermore, [30] traced the visual acuity in birds to the large cranial volume. These findings are confirmed in the present study in E. albus, as well as in other domestic ibis [31]
Magnification of the stratification traits in the epithelial layer of B. buteo can be traced to the reduction of light scattering and corneal opacity, besides the improvement of corneal transparency. Therefore, the epithelium of birds of B. buteo that inhabit high temperature desert areas represented highly refractive power lens for enhancement of its visual acuity and formation of sharp retinal image. These findings are in agreement with that observed in other predators [32, 33]
The recorded high variability in the stratified epithelium layer provides a protective layer against sever terrestrial surrounding habitats. Comparability of these observations are recorded in pensions and albatross [30, 34]. The present data are in disagreement with the findings of other studies conducted on other avian species and the Redtail Hawk (Buteo jamaicensis) [35,36,37].
Epithelium constitutes an effective power lens in the aquatic habitat, and a protective shield in the terrestrial habitat. Like many diver birds which have water accommodation range that enables them to control the corneal curvature and change it according to the surrounding habitats [28]. The greater curvature, high refractive power and increasing intensity of light enable them to overcome the variety of refractive indices of the aquatic optical system [28].
Regarding classic H & E investigations, the second layer of stroma illustrats the normal distribution of collagen lamellae. In case of E. albus and A.platyrhuncus, the collagen fibrils in their stroma condense as only one lamellar zone and have relatively small interfibrillar spaces. Such structure is considered the basic resistance to intraocular pressure, bears the tensile forces of the external environmental conditions, and protects intraocular tissues from trauma [38]. Similar findings are reached in Gallus gallus [39], and some vertebrates [40, 41].
Concerning Buteo buteo stroma, the present data distinguished it into two main bulks; loosed inner and compacted outer lamellar zones. The outer zone is perforated anteriorly by mostly circular interfibrillar space like a large entrance for the light beam that allows increasing the intensity of the light required for sharp retinal image and enhancement of the corneal transparency. Harsh habitats of these predators and their necessities for accurate vision to catch up their prey stimulate its morphological ultrastructure to adapt with their ecological surroundings [30].
As a result of these modifications, the collagen lamellae enables cornea to rotate their globes in a stable way and provides a clear protective goggle [42]. Previous studies, e.g. Boyce, Jones [43] and Neagu and Petraru [28], have revealed that the preying mechanism is based on the vision properties and the anatomical structure of eyes of both predators and preys. Whereas the predators possessed large eyes with more curved cornea, the preys have laterally small eyes and flattened cornea.
[38] proposed that the compacted lamellae of the stroma have a tendency to corneal opacity and cloudy properties, due to the low intensity of light passing through the narrow interfibrillar spaces which results in not frequently formation of sharp retinal image. Similar observations are made in the present study, whereas the corneal transparency of B. buteo is higher than that of the other two studied species, and are in agreement with the findings in Peregrine falcon (Falco peregrinus) and Redtail Hawk (Buteo jamaicensis) [37].
Accessory of the stromal layers of the three investigated species is formed from the polysaccharides ground matrix and proteoglycans producers (Keratocytes). They are distributed with moderate density in the two studied species E. albus and A. platyrhncus. Despite being extensively dispersed in the stroma of B. buteo especially, the anterior portion of the outer lamellar zone is aligned along its collagen lamellae. These observations are in agreement with Tsukahara, Tani [39] and Akhtar, Khan [44] in various species of domestic birds. Moreover, these observations illustrate the integrated function of the keratocytes as an enhancer for corneal transparency. Both Inoue, Watanabe [45] and Meek and Knupp [38] indicated that the light intensity strongly correlated with intensity of collagen in the stroma and stromal thickness in birds requiring high visual acuity and also in a number of proteoglycans. Therefore, keratocytes form more than 15% of corneal transparency [46,47,48].
The present histological demonstrations revealed the presence of two basal lamina (bowman's and Descemet’s membranes) in A. platyrhunicus and E. albus, limiting the two cellular layers epithelium and endothelium. In spite of their absence in the case of B. buteo cornea, they are well developed and identified in domestic birds, e.g. pigeon,penguins and albatross (aquatic aves) [28].
Generally, in various vertebrate groups, Bowman's and Descemet’s membranes contribute morphologically with less than 4% to the total corneal thickness and functionally with less than 30% to the corneal transparency [49]. Both of the two limiting membranes appeared in continuous state without any detectable interpretations or abnormalities in the investigated cornea [50]. Their homogenous elastic fibers are correlated to their cellular layers such as Struthio camelus and Dromaius novaehollandiae [51].
Bowman's membranes not only protect the inertocular layers from the harmful UV radiations, but also they protect producers of ground matrix of the stromal layers [44]. Recently, many authors reported that both bowman's and endothelium have high absorption coefficient compared to that of the stromal layer, which exceeds 30% of the total percent of absorption [52]. These could be due to the especially molecular composition and absorption coefficient of bowman's membrane and the elevated ratio of ascorbic acid content of epithelium layer, both of them acted as effective filter to protect the ocular tissue from harmful UV- radiation [44, 53].
However, absorption coefficient of stromal layer referred to the higher thickness and large polysaccharide content in their extra cellular matrix, besides microstructural organization of collagen fibers in Eolophus roseicapillus [54] and Bubo strix [55]. In case of absence of bowman’s membrane from corneal ultrastructure, as in B. buteo, stromal collagen fibrils are affirmably packed and are characterized by thin diameter to act as effective filter for absorption of the excessive UV spectra.
Generally, posterior basement membrane (Desecmet’s membrane) is considered a secretory product of endothelium layers during pre and postnatal stages as it is thickening with age. Chen, Li [56] reported that Desecmet’s membrane is constituted from different sorts of collagen fibrils and organized glycoprotein. They added that such posterior basement membrane is responsible for preserving the phenotypic and morphometric features of endothelium, besides maintaining the endothelium function under normal physiological conditions. Furthermore, Desecmet’s membrane provides anchoring to the posterior corneal surface during its morphogenesis process through acting as a stable scaffold (Chen et al. 2017). Akhtar, Khan [44] proposed that the thickness of Desecmet’s membrane does not exceed 0.5% of the total thickness, in spite of its vital role in preserving arid surrounding conditions on the stromal layer to prevent the corneal opacity. Moreover, this membrane has a regenerator property for the endothelium layer under mechanical scraping or wounding this layer in different animals [56].
The present study documented regular arrangement of the endothelium layer as single row of flat squamous cells at the separating boundary between corneal stroma and aqueous humor of the anterior chamber in the three studied species. Relatively, the same arrangement was assumed in penguin (Spheniscus magellanicus), Gallus gallus and Struthio camelus [55].
Pigatto, Laus [55] suggested that the endothelium layer is considered as a generator for most corneal tasks, such as declining stromal hydration, preserving corneal thickness, and improvement of corneal transparency. In addition to the small thickness of the endothelial layer in most vertebrates, especially birds, that does not exceed 5% of the total thickness, it works as very active pump of inflow water from aqueous humor and stromal matrix.
Moreover, thickness of corneal layers of the examined avian species recorded very high statistical significant variance (P < 0.000), where B. buteo (predators investigated species) demonstrated the higher thickness of the epithelial, stroma and endothelium layers, compared to the other two investigated species. Saadi-Brenkia, Hanniche [41] illustrated that higher thickness of stromal lamellae and corneal layers improve the corneal transparency and increase the corneal curvature. Such adaptations, according to the authors, are due to their functional transparency for the high temperature in arid habitat to catch preys.
A sharp retinal image stimulating excellent visual acuity, that is required for predator birds, can result from an enhanced corneal transparency and enlargement of light entrance. This can be achieved through increasing the thickness of corneal epithelium, protective google of the cornea and minimal stratification character [57]. Previous data demonstrated similar findings in Redtail Hawk Buteo jamaicensis and Peregrine falcon Falco peregrinus [37]. Additionally, high stromal thickness may be traced to large interfibrillar spaces between collagen lamellae, whereas it is responsible for increasing the quantity of incoming light and, therefore, declining the corneal opacity and light scattering that result from alteration of refractive indices of various visual habitats, as in falcons [37, 58].
Interestingly, species of intermediated habitat (amphibious vision), such as A. platyrhunces, possess relatively thinner stroma than the other two studied species. This can be traced to the high variability of their inhabiting conditions and their refractive indices of different optical medium. For these reasons, the thinner stroma has great impact on adaptation in various optical systems, which in turn increases corneal curvature and improves corneal transparency, as in albatross and penguin which are characterized by emmetropic cornea in air and water [59, 60].
Higher thickness of corneal endothelium maintains the phenotypic morphological composition and declines stromal hydration, as confirmed in various studies of pigeon and other species of birds [4].
Considering applying PAS stain on avian cornea, glycogen content is distributed relative to the biological requirements of the corneal layers for accommodation with different terrestrial, either forested or arid, habitat. The superficial corneal layer (epithelium) is loaded with variable degrees of PAS stain among the examined species; where it is strongly stained in E. albus; moderately stained in B. buteo, and weakly stained in A. platyrhunces. High glycogen content could be traced to the high metabolic activity and aerobic glycolysis of epithelial layer that result from biochemical reaction and oxygen consumption in different ecological conditions. These findings are similar to that documented in chick and domestic pigeon by Albuquerque, Pigatto [61].
The present study demonstrated these results in E. albus and its biological requirements to elevate the ratio of polysaccharides content and ensure its oxygen consumptions in case of the hydration state of stromal layer [8]. Previous studies reached similar findings in Gallus gallus domesticus and widespread species of birds [55].
The highly stained PAS cornea in the studied epithelial layer could be traced to the high absorption coefficient of UV- radiation. Exposure to UV radiation stimulates increasing the concentration of glycogen and glucose in the concerned tissue [26].
Moreover, moderated stained epithelium in B. Buteo revealed a high level of glycogen as a result of exposure to excess of UV spectra in their arid environment. Subsequently, low affinity of PAS reaction in the epithelium of A. platyrhunces came in agreement with the findings of previous researches conduced on sea otter, penguin and some species of falcons [4, 26, 28, 37].
The examined stroma of the three studied species recorded a strong affinity to PAS reaction in the outer lamellar zone of B. buteo and A. platyrhunces, but was weakly stained in E. albus. This may be due to the metabolic activity of the two examined species B. buteo and A. platyrhunce than E. albus, because of their body weight. The latter species raise their metabolic glycolysis and also their glycogen content as previously recorded in falcons and domestic pigeon [41]. In addition, B. buteo is characterized by higher daily activity, including catching preys and travelling long distances with high speed [30]. Therefore, their metabolic activity is higher and their glycogen composition is more elevated than other E. albus species.
The endothelium layer was recognized in the three studied species with strong affinity of PAS stain. This could be due to its cellular activity and higher oxygen consumptions that in turn stimulate concentration of polysaccharides [8]. Additionally, bowman’s and Descemet’s membrane showed high polysaccharides content in A. platyrhunces with amphibious visual characters. The absorption coefficient of UV radiation of these basement membranes is mailnly responsible for increasing glycogen composition. These cellular layers preserve the corneal stiffness and resist any tensile forces in most amniotes [62].
The investigated avian epithelial cell density showed very high statistically significant variations (P < 0.000), where great significance between B. buteo and A. platyrhunces is observed. Obviously, there is sharp decreasing in epithelial cell density in A. platyrhunces which may be due to absence of the role of corneal adaptation and activated lenticular accommodation. In case of submerged eye, such as that of A. platyrhunces in our investigation and that of cormorants [59], it is suggested that the lens is stimulated as an accommodative agent to adjust proper retinal image and compensate for the corneal transparency loss that results from similarity of refractive indices in aqueous humor and water.
Another critical point concerned the high epithelial cell density of B. buteo which could be due to its critical requirements for sharp retinal image and high visual acuity. Therefore, they are characterized by emmetropic eye in the terrestrial habitat and hyperopic in air to adapt to travelling large distances with high speeds and enhancement of its catching preys capacity. Relative significance of the cell density in epithelial layers was demonstrated in Chicken (Gallus gallus), Redtail Hawk (Buteo jamaicensis) and Peregrine falcon (Falco peregrinus) [37]. On the contrary, the flattened corneal species suffer from relative loss of corneal transparency during submerging and equality of refractive indices in cornea and water. Subsequently, the adaptive structural modification of their optical system stimulated their lenticular accommodation, whereas the optical lens compensated the corneal transparency loss. Such findings are equally suggested in penguins, albatrosses and seals [59].
Organizations of surface epithelial cells of avian corneal surface are characterized by highly regular polygonal epithelial cells in A. platyrhunces and sharp hexagonal cells with elevated borders and concentric notable nucleus in B. buteo. The high regularity of epithelial cells protects from the hydration status of the stromal layer, and ensures declining wet table epithelium and hydrated stroma [21, 63]. Some species of aves, like Phoenicopterus chilensis, Eolophus roseicapillus, Australian galah and Struthio camelus, exhibit the same regularity of polygonal cells mixed with hexagonal cells [10, 64]
The corneal surface microprojections, like microvilli, are elongated in B. buteo and shorted in A. platyrhunces. Microvilli contribute to 50% of the corneal transparency and are necessary for stabilization of tear film via adsorption of more mucin. This role coats and protects against infections from the surrounding habitat, as in the case of Dromaius novaehollandiae and Eudyptala [21]. Partially, sea otter and Penguins, as aquatic species, exhibited the same shorted microvilli that support nutritional exchanges [65].
The spreading of microholes over their corneal surface showed a high statistical significance in A. platyrhunces among the examined species. Dispersion of microholes over the corneal surface of the investigated species of birds is responsible for lubrication of the outer corneal surface and reducing the friction resistance that results from rapid movement of ocular adnexa [66].
Diffusion of cilia structure over the corneal surface of E. albus isolated the morphological features of epithelial cells and packed densely over the surface. Primary cilium represented microtubules that arise from inside organelle and protrude outside of plasma membrane and are responsible for morphogenesis and organization of corneal epithelial. On the other hand, cilia were represented rarely over the corneal surface of A. platyrhaunces. Surprisingly, the packing of cilia over corneal surface strongly correlated with increasing the cell density and enhancement of corneal transparency, according to Grisanti, Revenkova [67].