Ocular Morphology and Clinical Ophthalmic Parameters of Nile Tilapia Oreochromis niloticus
Dandara Franco Ferreira da Silva, Lucélia Gonçalves Vieira, Pedro Vale de Azevedo Brito, Sonia Nair Báo, Ingrid Gracielle Martins da Silva, Rodrigo Diana Navarro, Clarissa Machado de Carvalho, Paula Diniz Galera, Daniel Henrique Viana da Silva, Weslley de Souza Barbosa
TL;DR
This study describes the eye structure and establishes clinical reference standards for Nile tilapia using various imaging techniques.
Contribution
The study provides the first detailed ocular morphology and clinical ophthalmic reference data for Nile tilapia.
Findings
Nile tilapia have a scleral ring composed of cartilaginous and bony ossicles.
Clinical evaluations showed no ocular lesions with a mean intraocular pressure of 7.74 mmHg.
Ultrasonography and computed tomography effectively assessed ocular and retrobulbar structures.
Abstract
To describe the ocular morphological characteristics and establish clinical and ophthalmic reference standards for Nile tilapia. Fresh carcasses were used for diaphanization, optical and electron microscopy, and computed tomography analyses. For clinical and ultrasonographic evaluations, 75 adult Nile tilapias of undetermined sex were examined. Standard diaphanization, optical and electron microscopy, and computed tomography techniques were applied to fresh carcasses of Nile tilapia. Live fish were chemically restrained using propofol. Ophthalmic evaluations included slit‐lamp biomicroscopy, direct ophthalmoscopy, fluorescein staining, and rebound tonometry for intraocular pressure measurement. Ocular ultrasonographic measurements were obtained from both eyes of three individuals. The diaphanization technique revealed a scleral ring composed of two cartilaginous ossicles (dorsal and…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
FIGURE 1
FIGURE 2
FIGURE 3
FIGURE 4
FIGURE 5
FIGURE 6
FIGURE 7
FIGURE 8
FIGURE 9
FIGURE 10
FIGURE 11
FIGURE 12
FIGURE 13
FIGURE 14| Mean | SD | Median | Min | Max | IQR | |
|---|---|---|---|---|---|---|
| HH (cm) | 8.85 | 1.08 | 9 | 6.5 | 11.5 | 8.0–9.5 |
| BH (cm) | 12.17 | 1.43 | 12.25 | 8 | 15 | 11.5–13.0 |
| HL (cm) | 10.13 | 1.09 | 10 | 7.50 | 13 | 9.5–10.5 |
| SL (cm) | 31.43 | 2.71 | 31.5 | 24.5 | 37 | 29.5–33.0 |
| TL (cm) | 37.28 | 3.23 | 37.5 | 30 | 44 | 35.0–39.31 |
| EH right (cm) | 1.6 | 0.08 | 1.6 | 1.2 | 1.8 | 1.6–1.8 |
| EH left (cm) | 1.63 | 0.13 | 1.65 | 1.2 | 1.9 | 1.6–1.8 |
| EL right (cm) | 1.7 | 0.99 | 1.7 | 1.45 | 1.9 | 1.5–1.7 |
| EL left (cm) | 1.7 | 1.11 | 1.7 | 1.4 | 1.9 | 1.55–1.7 |
| Mean | SD | Median | Min | Max | IQR | |
|---|---|---|---|---|---|---|
| ALE (cm) | 1.05 | 0.06 | 1.08 | 0.97 | 1.11 | 1.01–1.1 |
| ACD (cm) | 0.10 | 0.02 | 0.10 | 0.08 | 0.13 | 0.09–0.11 |
| LENS (cm) | 0.28 | 0.01 | 0.29 | 0.27 | 0.30 | 0.28–0.29 |
| CORNEA (cm) | 0.05 | 0.01 | 0.05 | 0.04 | 0.07 | 0.04–0.05 |
| FPL (cm) | 0.62 | 0.06 | 0.62 | 0.54 | 0.71 | 0.58–0.66 |
| FPW (cm) | 0.2 | 0.02 | 0.19 | 0.18 | 0.24 | 0.18–0.2 |
| Mean | SD | Median | Min | Max | IQR | |
|---|---|---|---|---|---|---|
|
| ||||||
| DDEs (mm) | 7.63 | 1.63 | 7.9 | 4.32 | 9.76 | 7.37–8.44 |
| DDLs (mm) | 4.13 | 0.34 | 4.22 | 3.54 | 4.59 | 3.9–4.4 |
| RDEs (mm) | 70.6 | 20.3 | 83.2 | 45.9 | 97.7 | 50.3–86.5 |
| RDLs (mm) | 4.25 | 0.38 | 4.39 | 3.36 | 4.82 | 4.15–4.41 |
|
| ||||||
| LDEa (mm) | 67.6 | 14.1 | 70.2 | 42.7 | 86.1 | 63.5–77.3 |
| LDLa (mm) | 3.74 | 0.29 | 3.69 | 3.34 | 4.19 | 3.52–3.92 |
|
| ||||||
| LDEc (mm) | 5.82 | 1.55 | 5.26 | 4.60 | 8.94 | 4.79–5.94 |
| LDLc (mm) | 4.22 | 0.38 | 4.34 | 3.48 | 4.69 | 3.96–4.49 |
| RDEc (mm) | 13.2 | 2.3 | 13.5 | 8.9 | 17.1 | 11.4–14.6 |
| RDLc (mm) | 7.66 | 1.42 | 7.94 | 4.88 | 9.12 | 7.33–8.52 |
- —Coordenação de Aperfeiçoamento de Pessoal de Nível Superior10.13039/501100002322
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsOcular Disorders and Treatments · Retinal Development and Disorders · Corneal Surgery and Treatments
Introduction
1
Teleost fishes exhibit unique ophthalmic features that are closely linked to their environmental conditions and feeding habits. The sclera contains an osteocartilaginous ring that helps maintain the eye's shape and enhances the structural integrity of the eyeball [1]. However, unlike birds and reptiles, this structure does not participate in visual accommodation. Additionally, the pupillary light reflex is generally absent in teleosts due to the lack of iris muscle fibers [2].
Most studies emphasize the similarities among the various structures comprising the fish eye; however, interspecific differences are noteworthy. For example [3], the vertically elongated pupil of black pacu ( Colossoma macropomum ) is associated with its behavior of entering flooded forests to feed on seeds and fruits that fall onto the water's surface. Therefore, understanding the morphological and functional characteristics of each species is essential for advancing the ophthalmology of ichthyofauna.
Previous studies on the ocular morphophysiology of Nile tilapia ( Oreochromis niloticus Linnaeus, 1758) have primarily focused on the retina. The retinal photoreceptor layer of this species comprises single and double cones, as well as single rods, with their distribution varying according to light exposure [4, 5]. The commercial importance of Nile tilapia highlights the need for effective health management systems [6]. Additionally, Nile tilapia is widely employed as a bioindicator in biomonitoring studies of water pollution in tropical regions [7].
Regarding clinical ophthalmic parameters, previous studies have established reference values for rebound tonometry in koi fish ( Cyprinus rubrofuscus ), zebrafish ( Danio rerio ), brook trout ( Salvelinus fontinalis ), and Oscar fish ( Astronotus ocellatus ) [8, 9, 10, 11]. The anterior segment of the eye was evaluated in zebrafish and black pacu using slit‐lamp biomicroscopy, revealing varying degrees of light scattering by the cornea and lens [3, 12]. Imaging techniques are also valuable diagnostic tools; for example, ocular ultrasonography was used to diagnose retrobulbar neoplasia in flowerhorns (Cichlasoma sp.) [13] and to assess choroidal degeneration in halibut ( Hippoglossus hippoglossus ) [14].
Given the limited information on ocular morphology and ophthalmic examinations of Nile tilapia, this study aimed to describe the species' ocular anatomy using bone and cartilage diaphanization, as well as optical and electron microscopy. Additionally, clinical ophthalmological and imaging diagnostic parameters were characterized for this species.
Methods
2
Study Design and Animals
2.1
For the bone and cartilage diaphanization techniques, as well as the microscopic and tomographic analysis of Nile tilapia eyes, fresh carcasses donated by a commercial slaughterhouse were used. For optical and electron microscopy, the eyes were carefully excised from the heads, and the retrobulbar fat was removed.
Clinical and ultrasonographic evaluations of the eyes were performed on animals from the Aquaculture Center of the University of Brasília between July and October 2023. The fish were fed daily a diet of floating commercial pellets. Tanks with a capacity of 200 L, each housing up to 20 fish, were supplied with water from an artesian well. Water quality parameters (total ammonia, pH, and dissolved oxygen) were monitored daily using commercial kits (Labcon). Prior to evaluation, fish were fasted for 24 h. They were captured from the breeding tanks using a fishing net and placed in a 10 L plastic container for transport.
The animals were then transferred to a 20 L anesthetic induction aquarium, where they were immersed for 5 min in a propofol bath (Propovan, Cristália) at a concentration of 0.8 mg/L [15]. The aquarium was equipped with constant aeration provided by an air compressor (Model ACQ‐001, 16 W, 25 L/min flow, Guangdong BOYU Group Co). After immersion, fish were manually moved to a trough with a grid base covered by wet towels. While out of the water, oxygenation and maintenance of chemical restraint were ensured via a cannula inserted into the animal's mouth. This cannula was connected to a pump (Sarlo Better, Mini Model, 170 L/h) that circulated water from the induction aquarium.
Diaphanization of Bones and Cartilage
2.2
Diaphanization and optical microscopy analyses were conducted at the Multidisciplinary Laboratory of Morphology and Ontogeny, Institute of Biological Sciences, at the Federal University of Goiás (UFG). Three carcasses (six eyes) underwent bone and cartilage diaphanization to visualize the sclerotic ring, using an adapted technique [16, 17]. For this procedure, samples were placed in 2% potassium hydroxide (KOH), followed by staining of the ossicles with Alizarin red S and the cartilage with Alcian blue.
Light Microscopy
2.3
For light microscopy evaluation, 10 eyes were sectioned into sagittal and coronal slices using a microtome. Samples underwent routine histological processing, including dehydration in ethyl alcohol, clearing in xylene, paraffin infiltration, embedding in metal molds, and microtomy with semi‐serial sections of 6 μm thickness [18]. The sections were stained with hematoxylin and eosin and Masson's trichrome, then examined under a light microscope [19].
Electron Microscopy
2.4
Electron microscopy analyses were performed at the Laboratory of Microscopy and Microanalysis, at the Institute of Biological Sciences, at the University of Brasília (UnB).
Scanning Electron Microscopy (SEM)
2.4.1
Two Nile tilapia eyes, sectioned into parasagittal slices, were used for scanning electron microscopy (SEM) analysis. After fixation in 10% formalin, samples underwent standard SEM processing [20], which included buffering in sodium cacodylate solution, post‐fixation in osmium tetroxide, serial dehydration in acetone, mounting on metal stubs, critical point drying (Bal‐Tec AG, CPD 030), and gold sputter coating. The samples were then examined using a scanning electron microscope (JSM Jeol 7000F).
Transmission Electron Microscopy (TEM)
2.4.2
For transmission electron microscopy (TEM) analysis, two eyes sectioned into parasagittal slices and fixed in 10% formalin were used. Samples underwent post‐fixation in potassium ferricyanide and osmium tetroxide solution, contrasted with uranyl acetate, followed by serial dehydration in acetone with agitation. The tissue was embedded in Spurr resin and polymerized in an oven at 152.6°F (67°C). Semi‐thin and ultrathin sections were prepared using an ultramicrotome (Leica EM UC7) and examined with a transmission electron microscope (JEM Jeol 1011) [21].
Ophthalmic Clinical Evaluation
2.5
For the clinical evaluation, 75 adult Nile tilapia of undetermined sex, with an average body weight of 0.872 kg (IQR: 0.634–0.905 kg), was examined. A slit lamp (Kowa SL‐15) was used to evaluate and describe the cornea, lens, and iris. The fluorescein test was performed on both eyes to detect corneal lesions, using commercial strips (DrogaVet Str‐01) diluted in 5 mL of saline solution.
A direct ophthalmoscope (Heine mini 2000) with 15 and 20 diopter lenses was used to examine the posterior segment of the eye. Intraocular pressure was then measured using a rebound tonometer (Tonovet Plus, ICare) set to “canine” mode, and an applanation tonometer (TonoPen XL, Reichert), with the fish held in ventral recumbency.
Finally, ocular biometry was performed using a digital caliper (MTX 150 mm), and body measurements (Figure 1) were obtained using tape measures. The recorded measurements included eye height (EH), eye length (EL), head height (HH), body height (BH), head length (HL), standard length (SL), and total length (TL) [22].
Measurement of intraocular pressure using a rebound tonometer.
Ophthalmic Imaging
2.6
Ultrasonography
2.6.1
Ocular ultrasonographic measurements were obtained from both eyes of 3 fishes. Horizontal axial sections included axial length of the eyeball (ALE), anterior chamber depth (ACD), falciform process length (FPL), and falciform process width (FPW) (Figure 2B). Also, lens thickness was evaluated (Figure 2C). Ultrasonographic examinations were conducted using a Z60 Vet device (Mindray) in B‐mode with a linear transducer operating at 5–10 MHz. To reduce friction and protect the cornea, a thick layer of water‐based lubricant was applied to the eyes (Figure 2A).
Ocular ultrasonography in Nile tilapia. (A) Positioning of the transducer in a horizontal axial section to obtain the measurements. (B) Ultrasonographic image showing axial globe length (orange line), corneal thickness (yellow line), anterior chamber depth (blue line), falciform process length (green line), and falciform process width (white line). (C) Ultrasonographic image showing lens thickness (white dotted line).
Computed Tomography (CT)
2.6.2
For computed tomography (CT) six heads from fresh adult tilapia carcasses, donated by a commercial slaughterhouse, were used. Images were acquired in the rostrocaudal direction using a 16‐slice Aquilion Start CT scanner (Canon). Axial images were obtained following a standard protocol: 120 kVp, 150 mAs, acquisition matrix of 1024, field of view of 40.00/230.00, pitch 0.813, gantry rotation time 0.23 s, and slice thickness 1.0 mm.
Computed tomography images were analyzed using the Ambra ProViewer software (Version 3.24.2.0, Intelerad) in sagittal, axial, and coronal sections. Ocular structures measurements were performed in duplicate by two independent evaluators, and the final values were calculated as the mean of the measurements. The parameters assessed in the sagittal section included: rostrocaudal distance of the eyeball (RDEs) and rostrocaudal distance of the lens (RDLs) (Figure 3A) and dorsoventral distance of the eyeball (DDEs) and dorsoventral distance of the lens (DDLs) (Figure 3B).
Sagittal CT scan of Nile tilapia. (A) Horizontal measurements of the rostrocaudal distance of the eyeball (RDEs) and the rostrocaudal distance of the lens (RDLs). (B) Vertical measurements of the dorsoventral distance of the eyeball (DDEs) and the dorsoventral distance of the lens (DDLs).
In the axial section (Figure 4A), the following measurements were obtained: lateromedial distance of the eyeball (LDEa) and lateromedial distance of the lens (LDLa). In the coronal section (Figure 4B,C), the measurements included: lateromedial distance of the eyeball (LDEc), lateromedial distance of the lens (LDLc), rostrocaudal distance of the eyeball (RDEc), and rostrocaudal distance of the lens (RDLc).
Computed tomography images in axial (A) and coronal (B and C) sections of Nile tilapia. (A) Axial measurements of the lateromedial distance of the eyeball (LDEa) and lateromedial distance of the lens (LDLa); (B) Transverse coronal measurements of the lateromedial distance of the eyeball (LDEc) and lateromedial distance of the lens (LDLc); (C) Longitudinal coronal measurements of the rostrocaudal distance of the eyeball (RDEc) and rostrocaudal distance of the lens (RDLc).
Statistical Analysis
2.6.3
Descriptive analysis of the quantitative data (intraocular pressure, ocular and body biometrics, and ultrasound and tomographic measurements) was conducted using Microsoft Excel 365 MSO. Normality was assessed using the Lilliefors test. As most variables did not meet the assumptions of normality, Kendall's rank correlation test was applied to evaluate the relationships between body mass and body biometrics with ocular biometrics and tonometry, using the R software [23]. Correlation coefficients were interpreted as follows: values in absolute magnitude < 0.30 were considered weak, values between 0.30 and 0.50 as moderate, and values > 0.70 as strong correlations [24].
Results
3
Diaphanization Technique
3.1
The diaphanization process facilitated the evaluation of the morphology of the scleral cartilages and the scleral ossicles (Figure 5).
Photomicrograph of the skeleton of Nile tilapia after bone and cartilage diaphanization. (A) Lateral view of the complete skeleton; (B) Lateral view of the scleral ring; (C) Ventral view of the scleral ring; (D) Caudal view of the scleral ring. OsC, scleral cartilage; OsO, scleral ossicle.
Light Microscopy
3.2
In the histological evaluation, three distinct ocular strata were identified in Nile tilapia. The outermost layer, the fibrous tunic, is composed of the sclera and cornea. The middle layer, the vascular tunic, comprises the iris and choroid. The innermost layer, the nervous tunic, is constituted by the retina.
Fibrous Tunic
3.2.1
The sclera of Nile tilapia is composed of loose connective tissue fibers, with part of its surface covered by the sclerotic ring (Figure 6). The limbus was observed at the junction between the sclera and the cornea.
Photomicrograph of the ocular fibrous tunic of Nile tilapia in coronal section. (A) Ring of hyaline cartilage embedded in the sclera (10× magnification). (B) Limbus (arrow) (4× magnification). Masson's trichrome staining.
Five distinct strata were identified in the cornea. The most superficial layer, the corneal epithelium, was composed of a thin layer of cuboidal cells (Figure 7A). Immediately beneath it, Bowman's layer consisted of connective fibers arranged in bundles, with occasional interspersed epithelial cells (Figure 7A). The stromal layer contained disorganized collagen fibers oriented parallel to the corneal surface (Figure 7A,B). Descemet's membrane appeared as a thick layer of connective tissue (Figure 7B). The deepest layer, the endothelium, comprised a single row of simple cells (Figure 7B).
Photomicrograph of the cornea in coronal section (A, B) of Nile tilapia. BC, Bowman's layer; CE, corneal epithelium; DM, Descemet's membrane; En, endothelium; St, stroma. 10× magnification. Masson's trichrome staining.
Vascular Tunic
3.2.2
Among the components of the vascular tunic, the iris was fully identified. In contrast, the choroid, argentea, and falciform process were not observed in the examined sections; only the choroidal network was visible. This network consisted of capillaries and connective fibers arranged in parallel, enclosed by non‐adherent connective tissue (Figure 8A). The iris was composed of a bundle of connective tissue projecting into the eyeball toward the lens, bordered at its rostral edge by a band of pigmented cells, likely melanocytes (Figure 8B). No ciliary body was detected.
Photomicrograph of the vascular tunic of Nile tilapia in coronal section. (A) Choroidal network with capillary vessels filled with red blood cells (arrows) (10× magnification, hematoxylin and eosin staining); B. iris (4× magnification, Masson's trichrome staining).
Nervous Tunic
3.2.3
The retina of Nile tilapia consisted of 10 distinct layers. From the most superficial to the deepest, the stratification was as follows: pigmented epithelium (PE), photoreceptor layer (PL), external limiting membrane (ELM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GAN), axon layer (AXO), and internal limiting membrane (ILM). The PE appeared as a layer of elongated cells exhibiting a dark brown color, attributed to melanin presence, while the PL comprised cones and rods. The ELM was overlain by the branches of photoreceptors along its length, whereas the ONL and INL were identified as groups of nuclei arranged in clusters, resembling bunches of grapes. The OPL and IPL appeared as horizontal bundles, parallel to the pigmented epithelium. The GAN and AXO consisted of nerve fibers, and ILM presented as a layer of disorganized connective fibers.
Electron Microscopy
3.3
Scanning Electron Microscopy
3.3.1
Scanning electron microscopy (SEM) revealed the corneal epithelium and stroma, the lens, the pigmented epithelium, and the retinal photoreceptors. The corneal epithelium exhibited disorganized epithelial cells, while the stroma consisted of overlapping layers of collagen fibers (Figure 9A). Although the lens capsule was ruptured during processing, both supporting structures (suspensory ligament and the lens retractor muscle) were observed; however, they could not be distinctly differentiated (Figure 9B).
Scanning electron photomicrograph of the eyeball of Nile tilapia. A. Corneal epithelium (star) and stroma (circle) at 130× magnification of the cornea; B. lens with supporting structures (white arrows) at 85× magnification.
Visualization of the retina revealed the pigmented epithelium, characterized by bundles arranged in parallel, as well as photoreceptors (Figure 10).
Scanning electron photomicrograph of the retinal pigment epithelium of Nile tilapia, showing photoreceptors (white arrows) (330× magnification).
Transmission Electron Microscopy
3.3.2
Transmission electron microscopy (TEM) revealed photoreceptors and cells from the retinal nuclear layer. Cellular distribution was disorganized, likely due to tissue processing and fixation, which prevented determination of the proportion of cones and rods. Photoreceptors appeared as elongated and rounded structures, featuring a cell body with a peripheral nucleus located basally. Cells of the nuclear layer were rounded, vesicle‐filled, and also exhibited a peripheral nucleus (Figure 11).
Transmission electron photomicrograph showing photoreceptors in the retina of Nile tilapia. (A) Basal portion of the photoreceptor with peripheral nucleus (star) and apical portion of the photoreceptor (white arrow) (1200× magnification). (B) Cell of the nuclear layer (1200× magnification).
Ophthalmic Clinical Evaluation
3.4
No mortalities occurred during the clinical evaluation of the fish, and the established chemical restraint protocol was effective and safe for the procedures performed. Slit lamp biomicroscopy of the ocular surface revealed iridescent pigments in the bulbar conjunctiva, as well as in the limbal and peripheral regions of the cornea (Figure 12A). Compared to other vertebrates, the anterior chamber appeared shallower.
Ophthalmic evaluation of Nile tilapia using a slit lamp biomicroscope. (A) Presence of iridescent pigments in the bulbar conjunctiva, limbal, and peripheral regions of the cornea. (B) Aphakic crescent (AC), lens (Le) and pupil (Pu).
The pupils were rounded (Figure 12A,B), and the pupillary reflex was absent. The lens appeared spherical and was clearly visible, displaying the aphakic crescent (Figure 12B), which corresponds to the space behind the pupil lacking lens tissue. The cortical fibers of the lens were also discernible.
No animal exhibited ulcerative lesions in the fluorescein test. Based on the author's observations, maintaining ocular hydration by regularly flushing the eyes with saline solution while the fish are out of the water is essential to prevent lesions and avoid dye retention in dry areas, which could lead to misinterpretation. Under direct ophthalmoscopy, the fundus of the Nile tilapia appeared atapetal, avascular, and whitish with a pearly coloration. In some specimens, the falciform process was observed. However, capturing images of the fundus was not possible due to the small size of the direct ophthalmoscope lenses. Additionally, medial rotation of the eyeballs impeded the evaluation of all fundus structures.
Measurement of intraocular pressure using the applanation tonometer was unsuccessful, indicating that the rebound tonometer is better suited for use in Nile tilapia. A critical consideration for the rebound method is that corneal dryness can complicate obtaining accurate readings; therefore, maintaining corneal hydration is essential. The mean intraocular pressure measured with the rebound tonometer was 7.74 ± 1.71 mmHg (median: 7 mmHg, range: 5–12 mmHg).
In the Kendall correlation analysis, no significant correlations were observed, with weak associations between tonometry and body measurements: weight (τ = −0.0274; p = 0.7485), TL (τ = −0.0098; p = 0.9098), SL (τ = −0.0625; p = 0.4729), HH (τ = 0.0076; p = 0.9318), HL (τ = 0.0025; p = 0.9772), BH (τ = 0.1904; p = 0.0349), EL (τ = −0.0564; p = 0.3932), and EH (τ = −0.0034; p = 0.958).
Biometric Analysis
3.5
Descriptive statistics for body and ocular biometrics are presented in Table 1.
No strong correlations were identified in Kendall correlation analysis. However, moderate correlations were observed between eye length and the following variables: TL (τ = 0.3428; p < 0.001), SL (τ = 0.3291; p = 0.0001), BH (τ = 0.30115; p = 0.0006), and EH (τ = 0.4887; p < 0.0001). Regarding eye height, moderate correlations were found with body mass (τ = 0.4222; p < 0.0001), TL (τ = 0.3462; p < 0.0001), SL (τ = 0.3557; p < 0.0001), and BH (τ = 0.3723; p < 0.0001).
Ophthalmic Clinical Evaluation
3.6
Ultrasonography
3.6.1
Ocular ultrasonographic measurements of Nile tilapia are summarized in Table 2.
The general morphology of the eyeball appeared flattened, enclosed by an irregular hyperechoic line and filled with anechoic content. The cornea was visualized as a thin hyperechoic layer, also flattened at the anterior pole of the globe. The anterior chamber appeared as a space containing hyperechoic content. The lens exhibited a discrete hypoechoic layer located posterior to the anterior chamber (Figure 13A). The vitreous chamber presented as a broad anechoic space, bordered anteriorly by the lens and posteriorly by the falciform process. A prominent hyperechoic region was identified posterior to the falciform process, corresponding to the abundant retrobulbar fat characteristic of the species (Figure 13B).
Ocular ultrasound evaluation of Nile tilapia. (A) Image showing the cornea (yellow asterisk), anterior chamber (red asterisk), lens (green asterisk), and vitreous chamber (white asterisk). Note the flattened shape of the eyeball. (B) Falciform process (yellow arrows).
Computed Tomography
3.6.2
CT scanning revealed the lateromedially flattened shape of the eyeballs of Nile tilapia, allowing visualization of the vitreous chamber and lens in all projections. The cornea and lens exhibited hyperattenuating density, while the vitreous chamber appeared hypoattenuating (Figure 14).
Computed tomography of the head of a Nile tilapia in axial (A), sagittal (B), and coronal (C) planes. Note the cornea and lens (red triangle) with hyperattenuating density, and the vitreous chamber (yellow asterisk) with hypoattenuating density.
The measurements obtained from the computed tomography of Nile tilapia across the different sections evaluated are presented in the Table 3.
Discussion
4
The sclerotic ring of Nile tilapia consisted of two ossicles joined by two cartilaginous segments, consistent with descriptions in other teleost fishes such as Atlantic salmon ( Salmo salar ) and zebrafish [25, 26]. However, a third bone segment has been reported in the rostromedial region of the sclera in West African Denticle herring ( Denticeps clupeoides ), known as the Di Dario ossicle [27]. Regarding the positioning of these osteocartilaginous structures, in Mexican blind cavefish ( Astyanax mexicanus ), in contrast to the present study, the scleral cartilages are positioned diagonally rather than horizontally [28].
Regarding light microscopy of the fibrous tunic, the sclerotic ring and the five layers of the cornea were clearly visible in the coronal section. Variations in the characterization of these components among studies are attributable to interspecific differences, as well as to the quality and type of histological processing employed. In black pacu [3], the presence of all five corneal layers has also been reported. Conversely, Descemet's membrane was absent in rabbit fish ( Siganus javus ) [29], while Bowman's membrane was not observed in turbot ( Scophthalmus maximus ) [30]. In both rabbit fish and turbot, the iris consisted of connective tissue and a layer of brownish to blackish pigments; however, muscle bundles present in these species were not observed in Nile tilapia [29, 30].
The choroidal network in the teleost fish examined in the present study appeared as a cluster of capillaries containing red blood cells and connective fibers, similar to observations in turbot [30]. In contrast, in black pacus, the capillary vessels were surrounded by adipose tissue [3], whereas in rabbit fish [29], the choroid could be subdivided into layers of capillary vessels and connective tissue, with the choriocapillary layer corresponding to the choroidal network observed in tilapia. The retina of Nile tilapia displayed 10 distinct layers and showed no evidence of a tapetum lucidum, consistent with findings in black pacu and turbot [3, 30]. Individual identification of cones and rods within the retinal photoreceptor layer was not possible in this study due to histological processing challenges associated with the large size of the tilapia eye. The scleral thickness may have hindered fixative penetration into internal structures, thereby compromising the quality of the technique for examining delicate features such as blood vessels, iridal musculature, and retinal details.
Although electron microscopy processing in the present study followed the technique previously described for Nile tilapia [4], it was not possible to visualize the retinal pigment epithelium, the complete photoreceptor layer, or the outer nuclear, outer plexiform, and inner layers. This discrepancy may be related to the larger size of the eyeballs in the fish examined here, likely attributable to their age, which may have influenced the tissue's fixation properties. In the present study, adult animals were used, whereas the age of the specimens in the previous study was not specified.
For the SEM technique, the findings in Nile tilapia were consistent with those reported for mullet (Chelon auratus), in which the corneal epithelium was composed of cells with indeterminate shapes and numerous microprojections on their external surface, along with retinal pigmented epithelium arranged in bundles [31]. In contrast, in sandlance ( Limnichthys fasciatus ), the corneal surface displayed a mosaic pattern with numerous microvilli [20]. The lens and its supporting structures, represented in this species by the suspensory ligament and retractor muscle, were described similarly in turbot [30].
Visualization of photoreceptors by TEM corroborated most previously published data on teleost fish. Single cones, double cones, and rods were identified in Nile tilapia and Lessepsian rabbit fish ( Siganus luridus ) [4, 32]. In contrast, in true sardinella ( Sardinella aurita ), long single cones, short single cones, double cones, and rods were observed, along with an outer nuclear layer composed of photoreceptor nuclei [21].
Ophthalmic evaluation of Nile tilapia using a slit lamp revealed pigmentation characteristics and allowed clear visualization of the anterior segment components, as previously reported in black pacu [3]. In smaller species such as zebrafish, however, this examination has shown minimal light scattering by the cornea and lens, making it difficult to evaluate individual structures [12]. No pupillary reflex was observed in the fish examined in this study, consistent with reports for most teleost species [2].
Although the literature recommends the use of an indirect ophthalmoscope for evaluating the ocular fundus in fish, few references are available [2]. In the present study, the assessment of the full extent of the tilapia fundus was hindered by the medial rotation of the eyeballs, which may have resulted from chemical restraint with propofol or from involuntary ocular movements, the latter reported as common in teleost fish [33]. Despite these limitations, the evaluation was still effective and underscores the need for further studies on the clinical ophthalmology of different ichthyofaunal species.
An average intraocular pressure of 7.74 mmHg (±1.71 mmHg, standard deviation) was recorded in Nile tilapia, comparable to the 8 mmHg reported in brook trout [8]. In zebrafish, however, the average intraocular pressure was higher, at 14 mmHg^7^, whereas in koi and Oscar fish it was below 5 mmHg [8, 11]. Variations in intraocular pressure are often linked to the biomechanical properties of the eye and corneal thickness [34]. In omnivorous species such as Nile tilapia, the absence of active hunting behavior may substantially reduce visual demands, potentially contributing to a lower intraocular pressure.
Although glaucoma in teleost fish has only been reported following induction in experimental models [35, 36], further research is needed to determine its prevalence and clinical significance. It is therefore recommended that rebound tonometry be incorporated into routine ophthalmic evaluations of teleost fish, as the applanation tonometer proved ineffective. This limitation is likely attributable to specific anatomical characteristics of the fish eye, such as a notably shallow anterior chamber compared with mammals, as previously described in koi [8]. A strong correlation between total body length and eye diameter has been documented in white catfish ( Cathorops spixii ), wingfin anchovy ( Pterengraulis atherinoides ), and Naso stardrum ( Stellifer naso ) during both juvenile and adult phases [37]. In contrast, in Nile tilapia, pacu ( Piaractus mesopotamicus ), and guppy ( Poecilia reticulata ), no significant correlation was found between eye length and either head length or body length [38, 39]. Similarly, in the present study, no strong correlations were observed between body mass or other biometric measurements and ocular biometrics in Nile tilapia. These findings suggest that ocular growth in Nile tilapia is independent of overall body growth, as previously proposed [40].
Although ocular ultrasonography is a noninvasive, cost‐effective diagnostic method that provides immediate results, the literature on morphological and biometric descriptions in teleost fish is limited, and no published data on Nile tilapia were available prior to this study. Therefore, the findings presented here are novel. Ultrasonographic evaluation confirmed the lateromedially flattened shape of the Nile tilapia eyeball, in contrast to the spherical shape reported in black pacu. In both species, the cornea appeared as a hyperechoic line at the superficial pole of the eyeball, the lens was visualized as a hypoechoic structure anterior to the vitreous chamber, and the vitreous chamber was observed as a wide anechoic area located centrally within the eyeball [3].
The use of CT and other advanced diagnostic techniques in teleost fish remains rare. A study on black pacu carcasses characterized the sclera, vitreous chamber, and lens, consistent with findings in Nile tilapia [41]. In gilthead seabream ( Sparus aurata ), microtomography was employed to assess eyeballs and surrounding structures in relation to body fat levels [42]. Similarly, microtomography in zebrafish enabled identification of retinal nuclear layers through three‐dimensional reconstructions [43]. While CT in previous studies, including the present one, was performed on cadavers, its clinical applicability in live, chemically restrained animals is emphasized for evaluating critical ocular structures and adjacent tissues, such as retrobulbar fat.
Conclusions
5
The findings from diaphanization techniques, optical microscopy, and electron microscopy in Nile tilapia enabled detailed descriptions of the histology and morphology of the main ocular structures, consistent with the literature on most teleost species. Qualitative descriptions from clinical and diagnostic imaging examinations were provided, which may aid in the diagnosis of ocular disorders. Ocular and body biometric data were characterized to serve as reference values for the species. The information presented in this study is novel and paves the way for future research.
Author Contributions
Pedro Vale de Azevedo Brito: resources, methodology. Sonia Nair Báo: supervision, methodology, resources. Clarissa Machado de Carvalho: conceptualization, data curation, methodology, resources.
Funding
This study was financed in part by the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES).
Ethics Statement
This study complies with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Institutional Animal Care and Use Committee of the University of Brasília.
Conflicts of Interest
The authors declare no conflicts of interest.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1D. Goldman , “Müller Glial Cell Reprogramming and Retina Regeneration,” Nature Reviews Neuroscience 15, no. 7 (2014): 431–442.24894585 10.1038/nrn 3723 PMC 4249724 · doi ↗ · pubmed ↗
- 2I. Jurk , “Ophthalmic Disease of Fish,” Veterinary Clinics of North America: Exotic Animal Practice 5, no. 2 (2002): 243–260.12170631 10.1016/s 1094-9194(02)00006-3 · doi ↗ · pubmed ↗
- 3K. A. Gustavsen , J. R. Paul‐Murphy , E. S. Webber , et al., “Ocular Anatomy of the Black Pacu (Colossoma macropomum): Gross, Histologic, and Diagnostic Imaging,” Veterinary Ophthalmology 21, no. 5 (2018): 507–515.29380518 10.1111/vop.12539 · doi ↗ · pubmed ↗
- 4C. R. Braekevelt , S. A. Smith , and B. J. Smith , “Photoreceptor Fine Structure in Oreochromis niloticus L. (Cichlidae; Teleostei) in Light‐ and Dark‐Adaptation,” Anatomical Record 252, no. 3 (1998): 453–461.9811223 10.1002/(SICI)1097-0185(199811)252:3<453::AID-AR 13>3.0.CO;2-3 · doi ↗ · pubmed ↗
- 5A. M. Azab , H. M. Shoman , R. M. El‐Deeb , H. M. Abdelhafez , and S. A. Samei , “Comparative Studies on the Histology of Eye Retina in Some Nile Fishes With Different Dial Activities,” Egyptian Journal of Hospital Medicine 68 (2017): 815–823.
- 6ABINPET. Brazilian Association of Pet Products , “2023 Market Data,” (São Paulo: ABINPET, 2023), https://abinpet.org.br/wpcontent/uploads/2023/03/abinpet_folder_dados_mercado_2023_draft 1_incompleto_web.pdf.
- 7F. Peixoto , D. Alves‐Fernandes , D. Santos , and A. Fontaínhas‐Fernandes , “Toxicological Effects of Oxyffuorfen on Oxidativestress Enzymes in Tilapia Oreochromis niloticus ,” Pesticide Biochemistry and Physiology Vila Real 85 (2006): 91–96.
- 8G. L. Lynch , A. Hoffman , and T. Blocker , “Central Corneal Thickness in Koi Fish: Effects of Age, Sex, Body Length, and Corneal Diameter,” Veterinary Ophthalmology 10, no. 4 (2007): 211–215.17565552 10.1111/j.1463-5224.2007.00538.x · doi ↗ · pubmed ↗