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Structure and microanalysis of tear film ferning of camel tears, human tears, and Refresh Plus

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Purpose: This study aimed to investigate the tear ferning pattern and chemical elements of the tear film of camel tears compared with human tears and Refresh Plus eye lubricant. Refresh Plus was used as a control because it provides a healthy ferning pattern, due to the presence of an optimum ratio of carboxymethylcellulose (CMC) sodium and electrolytes. The main research focus is elucidating the viability of camel tear film in the dry, harsh environment of the desert. Methods: The tears were collected from five camels, five male desert workers (20-25 years old) at a small village located 100 km from Riyadh, Saudi Arabia, and five male subjects (20-25 years old) from Riyadh. A small drop (1 μl) of tears was dried on a glass slide and observed under a light (Olympus BX1) and scanning electron microscope (Inspect S50, Field Electron and Ion Company [FEI]). Energy-dispersive X-ray spectroscopy (EDS) of the tear film and Refresh Plus were investigated with a JEOL 1400 scanning transmission electron microscope. Results: The camel tear film pattern was surrounded by thick, peripheral, homogenous layers containing small oily droplets, particles, and tiny branches in the tear ferning. The tear ferning of the camel was grade 0-1, whereas the tear ferning of human tears and Refresh Plus was grade 1-2. The mass percentage of chloride was highest in the camel tears. The mass percentage of potassium in the camel tears was greater than that in the human tears, but it was less than that in the Refresh Plus lubricant. Conclusions: Camel tears exhibit a better quality than human tears and Refresh Plus lubricant do. The presence of oily droplet-like structures at the periphery of tear ferning suggests that camel tear film may have a higher quality and quantity of minerals and lubricants, which may help the animal to avoid eye dryness. Future work is required to investigate the identification of the elements present in the peripheral and central part of the tear ferning.
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The dromedary camel (Camelus dromedarius) is a native
of the dry, sandy climate of Saudi Arabia. In these harsh
climatic conditions, camel eyes are wet and full of tears. The
tear film is a thin layer that covers the anterior part of the eye.
Its presence is important for maintaining the ocular surface
and providing a defense against pathogens and foreign bodies
[1]. The ocular tear film can be classified into three layers,
as follows: the outer lipid layer, intermediate aqueous layer,
and underlying mucus layer [2,3]. The loss of the quality or
quantity of tear film can result in dry eye disease [3].
The ferning phenomenon is a dendritic growth patter n of
dried tear fluid. It is influenced by the secretions’ electrolyte,
protein, and mucus contents. When mucus is permitted to
air dry on a microscope slide, a specific type of crystalli-
zation called a fern occurs, and this phenomenon has been
termed “ferning.” The tear ferning test has been described as
a simple test that can be used to evaluate tear film [4] and has
the potential to be applied in clinical practice [3].
Rolando suggested a grading scale for tear ferning. Types
I and II indicate normal tear film, while types III and IV
indicate dry eye [5]. Recently, Masmali et al. [6] developed
a new tear ferning grading scale, where grade ≥ 2 indicates
dry eye. The application of the Masmali tear ferning grading
scale has shown that the tear ferning test has good validity
and reliability, and there is no change in ferning patter ns
during the day [6]. The tear ferning test has the right features
for use in the eye clinic for dry eye diagnosis [7-9]. Pearce
and Tomlinson [10] studied tear film ferning with light and
electron microscopy, and they discussed the role of chemical
elements, such as sodium (Na), potassium (K), chlorine (Cl),
and sulfur (S), in the formation of ferning.
Few studies have performed tear ferning tests on the
tear film of animals and compared their patterns with that
of humans. Silav et al. [7] investigated the tear ferning of
healthy horses and found Types I, II, and III ferning in these
animals. The authors counted the points on the crystallized
ferning pattern and correlated the pattern with other param-
eters to assess the ocular surface. According to these authors,
the tear ferning test is easy to perform and a good tool for
assessing tear film quality.
Molecul ar Visi on 2018; 24:305-314 <http://www.molvis.org/molvis/v24/305>
Received 8 November 2017 | Accepted 13 April 2018 | Published 16 April 2018
© 2018 Molecular Vision
305
Structure and microanalysis of tear lm ferning of camel tears,
human tears, and Refresh Plus
Masmali AM,1 Fagehi RA,1 Ahmad H. El-Naggar,2 Almubrad TM,1 Akhtar S1
1Cornea Research Chair, Department of Optometry, College of Applied Medical Science, King Saud University, Riyadh, Saudi
Arabia; 2Soil Sciences Department, College of Food & Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia
Purpose: This study aimed to investigate the tear ferning pattern and chemical elements of the tear lm of camel tears
compared with human tears and Refresh Plus eye lubricant. Refresh Plus was used as a control because it provides a
healthy ferning pattern, due to the presence of an optimum ratio of carboxymethylcellulose (CMC) sodium and electro-
lytes. The main research focus is elucidating the viability of camel tear lm in the dry, harsh environment of the desert.
Methods: The tears were collected from ve camels, ve male desert workers (20–25 yea rs old) at a small village locate d
100 km from Riyadh, Saudi Arabia, and ve male subjects (20–25 years old) from Riyadh. A small drop (1 μl) of tears
was dried on a glass slide and observed under a light (Olympus BX1) and scanning electron microscope (Inspect S50,
Field Electron and Ion Company [FEI]). Energy-dispersive X-ray spectroscopy (EDS) of the tear lm and Refresh Plus
were investigated with a JEOL 1400 scanning transmission electron microscope.
Results: The camel tear lm pattern was surrounded by thick, peripheral, homogenous layers containing small oily
droplets, particles, and tiny branches in the tear ferning. The tear ferning of the camel was grade 0 –1, whereas the tear
ferning of human tears and Refresh Plus was grade 1–2. The mass percentage of chloride was highest in the camel tears.
The mass percentage of potassium in the camel tears was greater than that in the human tears, but it was less than that
in the Refresh Plus lubricant.
Conclusions: Camel tears exhibit a better quality than human tears and Refresh Plus lubricant do. The presence of oily
droplet-li ke st ruct ures at the per ipher y of tear fe rning suggests that camel tear l m may have a higher quality and quan-
tity of minerals and lubricants, which may help the animal to avoid eye dryness. Future work is required to investigate
the identication of the elements present in the peripheral and central part of the tear ferning.
Correspondence to: Saeed Akhtar, Cornea Research Chair,
Department of Optometry, College of Applied Medical Science,
King Saud University, PO Box 10219, Riyadh, Riyadh 11433, Saudi
Arabia; Phone: 00 966 54 103 2226; FAX: 0096 6114693536; email:
Akhtars@ksu.edu.sa
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306
In the present study, we investigated the quality of
camels’ tear film using tear ferning techniques and conducted
a comparison with Refresh Plus and the tear films of humans
living in desert and urban areas. “Refresh Plus” was used as a
control because it contains an optimum ratio of carboxymeth-
ylcellulose (CMC) sodium and electrolytes. The ratio between
electrolytes and large molecules (CMC sodium) could be the
main reason for its production of healthy ferning patterns
[11]. The present study also aims to elucidate the viability
of the tear film in the dry, harsh environment of the desert.
The chemical element analysis of the tear film ferning was
performed using scanning transmission electron microscopy
(STE M).
METHODS
The study was approved by The College of Applied Medical
Sciences (CAMS) Ethical Committees of King Saud Univer-
sity, Riyadh, Saudi Arabia (ethics number: CAMS 53–35/36).
The tear film pattern was investigated using a light and scan-
ning electron microscope. Tears were collected from five
camels (males) and five desert workers (males, 20–25 years
old) from a small village located 100 km from Riyadh, Saudi
Arabia. Tears were also collected from five male subjects
(20–25 years old) living in Riyadh. The human subjects
underwent ophthalmic evaluation to confirm that the ocular
surfaces of their eyes were healthy. The human and camel
tears were collected in the same way using a microcapillary
tube (Drummond Scientific Co., Broomall, PA). Refresh Plus
(Allergan) was bought from a local pharmacy shop. The main
constituents of Refresh Plus are CMC sodium (0.5%), calcium
chloride, magnesium chloride, potassium chloride, water,
sodium chloride, sodium lactate, and hydrochloric acid.
A small drop (1 μl) of each of the different types of tears
and the artificial tears (Refresh Plus, Allergan) was placed
on a glass slide at 23 °C temperature and relative humidity
of less than 45%. When the tears were dried (3 h after collec-
tion), they were observed under a light microscope (Olympus
BX1) and classified according to the Masmali (2015) scale
[10]. The Masmali grading scale for tear ferning was used to
grade the ferning patterns.
The tear film and Refresh Plus ferning patterns were
observed using an Inspect S50 environmental scanning elec-
tron microscope. For scanning microscopy, 1-μl drops of each
tear type and Refresh Plus were placed on a small piece of
glass. This glass piece containing the tear film droplet was
stuck on the scanning electron microscope stub. The tear
film was gold coated and observed under an Inspect S50,
FEI Quanta 200 environmental scanning electron micro-
scope. Digital images were captured using an xT microscope
Server software and processed with Scandium (Olympus Soft
Imaging Solutions GmbH).
The energy-dispersive X-ray spectroscopy (EDS) of the
tear film and Refresh Plus were performed using the JEOL
1400 STEM. The microscope was equipped with STEM
Viewer software and a Valita camera. Small droplets were
placed on formvar-coated, 200-mesh nickel grids and dried
for 30 min. Formvar is a thin, electron-transparent layer
coated on the grids. It is used to hold the sample in place
when conducting electron microscopy. It is routinely used
for liquid samples. The ferning pattern that formed on the
formvar-coated grids was observed using STEM and then
EDS element analysis. This was conducted at different
regions of ferning. The microanalysis of the elements was
done using the STEM Viewer software, and digital images
were taken with the Valita camera.
RESULTS
Light microscopy of ferning of camel, human, and Refresh
Plus tear film: Our light microscopy observations showed
that the ferning patterns at the periphery and center of the tear
film were extremely similar. The observation and imaging
of the ferning for the light microscopy was carried out from
the periphery to the center of the tear film. The peripheral
part of the camel tear film pattern was surrounded by five
layers (labeled 1, 2, 3, 4, and 5; Figure 1A,B). The first
layer (1) was pale in color and consisted of a homogenous
material, whereas the second layer (2) was dark brown and
contained small oily droplets (Figure 1A,B). The third layer
(3) was blueish, while the fourth layer (4) was grayish and
contained tiny droplets (Figure 1A,B). Finally, the fifth layer
(5) was light brown and contained droplet-like structures.
The ferning pattern was present in the fifth layer, with
tree-like branches with leaves present just inside this layer,
(Fig ure 1C,D). Inside these tree-like branches, fern plant–like
branching was present. Numerous secondary and tertiary
branches emerged from the primary branches (Figure 1E,F).
The peripheral and central ferning patterns of the camel tear
film were extremely similar, containing dense, thin branches
(Figure 1C-F). According to the Masmali grading scale, the
tear ferning at the periphery and center could be categorized
as grades 0–1 (Figure 1C-F).
The ferning patter ns of the humans residing in the urban
environment and the desert were similar. In both cases, the
peripheral part of the tear film pattern was surrounded
by three layers (1, 2, and 3; Figure 2A-C). The first layer
consisted of a whitish homogenous material, while the second
layer was made up of dense granular material (Figure 2C).
Finally, the third layer contained fine, small ferning (Figure
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Figure 1. Light micrographs of the peripheral and central ferning pattern of camel tear lm. A and B: Part of the peripher y of the tear lm
show ing the ve outermost layers: 1) cre am-colored homogenous layer, 2) dark brow n oily droplet layer, 3 and 4) smal l drople t laye rs, 5) th ick
ferning pattern layer. C: Part of the periphery of the tear lm showing a dense ferning pattern below the peripheral layers. D, E, F: Part of
the center of the tear lm showing prominent primary, secondary and tertiary branching. 1 = First layer, 2 = Second layer, 3 = Third layer,
4 = Fourth layer, 5 = Fifth layer, D = Oily droplets, F = Ferning, PF = Primary ferning, SF = Secondary ferning, TR F = Tertiary ferning.
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Figure 2. Light micrographs of the peripheral and central ferning patterns of desert human tear lm. A, B: Peripheral part of human tear lm
showing peripheral ferning surrounded by three layers: 1) white homogenous layer, 2) dark brown homogenous layer, 3) granular layer with
network like structure. C: “Tree branch” ferning pat tern at the periphery of tear lm below the peripheral layers. D, E, F: Ferning pattern
of the center of the human tear lm showing prominent primary, secondary, and tertiary branching. 1 = First layer, 2 = Second layer, 3 =
Third layer, F = Ferning, PF = Primary ferning, SF = Secondary ferning, TRF = Tertiary ferning.
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Figure 3. Light micrographs of the peripheral and central ferning patterns of Refresh Plus. A , B, C: Peripheral part of Refresh Plus surrounded
by four layers: 1) outermost layer, white in color and homogenous; 2) granular white layer; 3) thick granular layer; 4) granular dark brown
layer. Just below the fourth peripheral layer, the ferning contains only primar y branches. Away from the periphery, the ferning pattern
has secondary and tertiary branching. D, E, F: Ferning pattern in the central part of the Refresh Plus lubricant containing secondary and
tertiary branching. 1 = First layer, 2 = Second layer, 3 = Third layer, 4 = Fourth layer, 5 = Fifth layer, F = Ferning, PF = Primary ferning,
SF = Secondar y ferning.
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2C). Just inside the third layer, the ferning was like the
branching of a tree, and this was different from the central
ferning (Figure 2C). The secondary and tertiary branches of
ferning shoots from the primary branches were dense and
thin (Figure 2C-F). According to the Masmali grading scale,
the ferning patter n in the center and periphery could be
categorized as grades 1–2 (Figure 2A-D).
In Refresh Plus, the ferning pattern was surrounded by
four layers (1, 2, 3, and 4; Figure 3A-C). The first, outer-
most layer (1) was a whitish homogenous layer, while the
Figure 4. Scanning electron micrographs of the ferning pattern of camel tear lm, human tear lm, and Refresh Plus lubricant. A, B: Part
of the camel tear lm showing primary (pf), secondary (sf), tertiary (trf), and ne ferning; C, D: Human tear lm containing primary (pf)
secondary ferning (sf) and ne fer ning. E , F: Refresh Plus ferning showing primary and secondary branching. 1 = First layer, 2 = Second
layer, 3 = Third layer, 4 = Four th layer, 5 = Fifth layer, CR = Crystals, PF = Primary fer ning, SF = Second ar y fern ing, TR F = Tertia ry ferning.
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second (2) and third (3) layers were granular (Figure 3A-C).
The fourth layer (4) was dark brown and formed a network
structure (Figure 3C). The ferning patterns in the periphery
and center were similar. The branching of the secondary and
tertiary branches was not clear and defined. The peripheral
and central ferning patterns were thick and belonged to grade
2 (Figure 3D,E,F).
Scanning microscopy of tear ferning of camel and human
tears and Refresh Plus: Scanning microscopy showed further
fine secondary and tertiary (offspring) branches in the tear
Figure 5. Energy-dispersive X-ray spectroscopy (EDS) analysis of dendrites in tear lm showing peaks of the mass percentage of chloride
and potassium in camels, humans, and Refresh Plus lubricant. The tear samples were placed on nickel grids. The unlabeled peaks are nickel
(Ni) from the nickel gr ids. The labeling of the peaks from backgrounds, such as nickel grids, were excluded. ClKa = Chlorine at Kα, FeKa
= Iron at Kα, FeLa = Iron at Lα, K = Potassium, KKa = Potassium at Kα, KKb = Potassium at Kβ.
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ferning of camel tears, human tears, and Refresh Plus (Figure
4A-F). The offshoots of branches of the ferning pattern in
the camel tears (Figure 4A,B) were thinner compared with
those in human tears (Figure 4C,D) and Refresh Plus (Figure
4E,F). There were tiny crystals between the branches of the
tear ferning (Figure 4A,B).
The microanalysis of the elements took place from the
periphery to the center of the tear film. The analysis of the
elements was performed at a different part of the ferning. The
EDS analysis showed that the mass percentage of chloride was
highest in the camel tear ferning compared with the human
tears and Refresh Plus (Figure 5). The mass percentage of
potassium in the camel tear ferning was greater than that in
the human tears, but it was less than that in the Refresh Plus
lubricant (Figure 5).
DISCUSSION
Our observations showed that the branching pattern in camel
tears was more prominent and dense than that in human
ferning, especially in the tertiary branches. The branching in
the outermost layer of the tear film was denser in camel than
human tears. Furthermore, there was a difference between
the central and peripheral ferning of the camel tear film. The
tear film ferning of humans living in the desert and city were
similar to each other.
In the camel, the ferning pattern of the tear film was
surrounded by five layers (1, 2, 3, 4, 5; Figure 1A), whereas
in human and Refresh Plus, it was protected by three and four
layers, respectively. It is documented that sulfur-containing
amino acid residues, namely cysteine and methionine, are
present in the proteins and protein backbone of mucin [10].
This leads us to think that macromolecules like mucin and
proteins are present at the periphery of tear ferning [10]. We
consider that the peripheral layers of the tear film ferning
were formed from the sulfur-containing proteins and mucin.
The camel is a native of dry, harsh weather conditions.
Its ability to withstand dry and semidry weather could be
due to the presence of antibody molecules that have only two
heavy chains, making them smaller and more durable [8].
Camel eyes are protected by two rows of eyelashes and three
eyelids, which provide protection from the sand and the harsh
weather conditions [9]. The camel’s cornea constitutes 35%
epithelium and 60% stroma [12], and the thick epithelium
protects the stroma from drying out in the arid weather [12].
To lubricate the thick epithelium of the cornea, the camel tear
film presumably has a special composition of proteins, lipids,
and minerals.
The abundance of proteins in the tears of humans and
other animals has been analyzed previously [13,14]. Ly so-
zymes were observed in the tears of humans and domestic
animals (sheep, goats, llamas, cattle, horses, dogs, and
ra bbits) [13,14]. Furthermore, the lysosomes in humans are
synthesized in the acinar cells of the lacrimal glands and
constitute 20– 40% of the total tear protein [15,16]; they
exhibit antibacterial activity and hydrolyze the glycosidic
bonds of certain Gram-positive bacteria [15,16].
Shamsi et al. [17] investigated tear proteins, namely lyso-
some, lactoferrin, lipocalin, and vitelline membrane outer
layer protein 1 (VMO1), in cow, sheep, human, and camel
tears. The authors reported that the cow and sheep tears had
a lower concentration of lysosomes compared with the human
and camel tears. The lipocalins were identified only in the
cow and sheep, whereas VMO1 was found in the sheep and
camel tears but not in the human and cow tears. Furthermore,
higher VOM1 levels were found in summer tears compared
with winter tears [18]. It has been suggested that VOM1
expression may serve to maintain the ocular surface by
keeping the tear film stable [19,20]. We think that VOM1 may
lubricate the cornea in the harsh and dry climate to protect it
from infection.
The camel tear film ferning was surrounded by thick
layers of homogenous material containing oily, droplet-like
structures. These structures were not observed in the human
tears or Refresh Plus. It has been reported that tears contain
phosphatidylcholines (PCs), sphingomyelins (SMs), wax
esters (WEs), and free cholesterols (Chos) [21]. In addition
to PCs and SMs, the presence of triacylglycerides (TAGs),
ceramides (Cers), and phosphatidylethanolamines (PEs) in
tears has also been observed [22]. Recently, Lam et al. [23]
reported the presence of a novel lipid amphiphile, cholesteryl
sulfate (CS), in human tears. We speculate that the oily drop-
lets present in the outer peripheral layer of the tear ferning
may be composed of the lipids present in the tears, inhibiting
evaporation, and therefore, dryness of the eye. The presence
of highly oily droplets may protect camel tears from drying
out in the hot, dr y climate.
The tear ferning pattern is thought to be formed due to
the concentration and interaction of electrolytes, especially
sodium and chloride, with macromolecules, such as tear film
mucins and proteins [24]. This concentration, especially the
ratio of monovalent Na and K to divalent Ca and Mn, is the
key to the formation of tear ferning [25]. Pearce et al. [10]
also suggested that the balance of Na, K, and Cl—rather
than the level of a single element—is responsible for the
ferning formation. The authors further suggested that mucin
and proteins are not part of the fern structure; instead, their
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prohibition from macromolecules helps the formation of
ferning to progress. Golding et al. [11] supported the view
that fern crystals are mainly composed of sodium chloride
and potassium chloride, without any participation of protein
and mucin, contradicting their [24] previous hypothesis.
The structure analysis showed that the camel tear ferning
was better than that of the Refresh Plus lubricant. The
scanning electron microscope showed that the tertiary and
quaternary branches were fine in the camel tears compared
with the human tears and Refresh Plus. These observations
suggest that the elements responsible for the ferning pattern
were more prevalent in the camel tears than in the human
tears and Refresh Plus. Our present study showed the pres-
ence of higher concentrations of chloride and potassium in
camel tear ferning. It is thought that Cl and K may facili-
tate the healthy formation of tear ferning in the camel. To
our knowledge, EDS results on Refresh Plus have not been
published. However, our elemental analysis showed the pres-
ence of elements (Cl, K) that were present in the Refresh Plus
ingredients in EDS.
The tear ferning of Refresh Plus was dense and thin
compared with that of the human tear film. The ingredients
reported in Refresh Plus are CMC sodium (0.5%), calcium
chloride, magnesium chloride, potassium chloride, purified
water, sodium chloride, and sodium lactate. We think that
the quantities of these components or their relationships are
greater in the camel tears compared with the Refresh Plus
and human tears, resulting in the higher grade of ferning
in camel tears. We speculate that the formation of the outer
oily droplets and homogenous layers in camel tear ferning
could be formed due to the presence of the higher content of
lipid, mucin, and multiple sulfur-containing proteins, such
as lactoferrin, lipocalin, and VOM1. Healthy tear film helps
camels to protect their eyes from dryness in the harsh, arid
climatic conditions. Future work is required to investigate
the identification of the droplet-like structure and chemical
elements present in the peripheral layers and central part of
the tear ferning.
ACKNOWLEDGMENTS
The project was supported By King Saud University, the
Deanship of Scientific Research, Research Chairs.
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Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China.
The print version of this article was created on 16 April 2018. This ref lects all typographical corrections and errata to the article
through that date. Details of any changes may be found in the online version of the article.
... 14 The TF test has good specificity and sensitivity and has been used with other tests to assess the quality of tears in both animals and humans. [19][20][21][22][23][24][25][26][27] Goats and camels have big eyes and are considered good animal models for surgical interventions. 28,29 Therefore, this study investigated the effect of adding electrolyte solutions on the TF patterns of tears collected from goats and camels. ...
... 33 Notably, the TF patterns of tears collected from camels outperformed the corresponding ones for Refresh Plus eye drops. 27 The scanning electron microscope revealed that the tears collected from camels have perfect tertiary and quaternary divisions. 27 The ions such as K + and Clare responsible for fern formation and were more prevalent in tears collected from camels compared with human tears and Refresh Plus eye drops. ...
... 27 The scanning electron microscope revealed that the tears collected from camels have perfect tertiary and quaternary divisions. 27 The ions such as K + and Clare responsible for fern formation and were more prevalent in tears collected from camels compared with human tears and Refresh Plus eye drops. 27 Presumably, these anions help maintain healthy TF patterns in camels. ...
Article
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Background. Good quality of tear film is essential for healthy vision in both animals and humans. Therefore, improving the quality of tears through the addition of electrolytes is important. Objectives. To assess the effect of adding various electrolyte solutions on tear ferning (TF) patterns collected from goats and camels. Materials and methods. Tear samples (20 μL) were collected from 5 goats (2 males and 3 females; 3.4 ±1.6 years) and 5 camels (2 males and 3 females; 4.0 ±1.1 years) using microcapillary tubes. A tear sample (0.5 μL) from each animal was mixed with various volumes (0.5–5 μL) of each electrolyte solution to produce homogenous mixtures. A sample (1 μL) of each mixture was dried on a microscopic glass at 22°C with a humidity ≤40%. The obtained TF pattern was observed, graded and compared with those obtained for the corresponding pure tear samples. The effect of dilution using purified water on the TF patterns of animals was also tested. Results. The TF grades of animals were generally enhanced when mixed with electrolyte solutions. Specifically, the TF grade for tears collected from a goat was improved from 1.4 to 0.7 and to 0.8 when magnesium chloride hexahydrate and calcium chloride were added, respectively. Similarly, the TF grade for tears collected from a camel was improved from 1.8 to 0.9 and to 1.1, when calcium chloride and sodium dihydrogen phosphate solutions were added, respectively. Conclusions. The TF grades of tears collected from both goats and camels were improved after adding electrolyte solutions, and they were most remarkably improved when divalent electrolyte solutions were added, followed by the hydrogenated electrolyte.
... The TF test was used to analyze the tears collected from camels and compare their compositions with those for artificial eyedrops and humans living in the desert of Saudi Arabia [56]. The grades of camels' tears ranged from grade 0 to 1 based on the five-point grading scale that indicated normal and healthy eyes. ...
... The grades of camels' tears ranged from grade 0 to 1 based on the five-point grading scale that indicated normal and healthy eyes. In contrast, the TF grades for human tears (N = 5) and Refresh Plus ranged from 1 to 2. [56]. Furthermore, they showed a higher chloride level compared to human and artificial tears. ...
... Furthermore, they showed a higher chloride level compared to human and artificial tears. The potassium level was higher in Refresh Plus eyedrops followed by camels and human tears [56]. Camels' tears contain higher levels of lubricants and mineral compared with those for artificial and human tears. ...
Article
Full-text available
Background: Tear film stability is essential for healthy vision. More than one test should be used to examine tear film status. The tear ferning test is used as a valuable and repeatable tool to examine the quality of tears in animals and human. The current study aimed to report and discuss the most recent use of the tear ferning test to diagnose dry eye in animals and human. Main text: The tear ferning test involves the collection of a tear sample using a capillary tube. The tears were allowed to dry in a controlled environment, and the patterns produced were observed and inspected using a light microscope. The four-point tear ferning grading scale that included type I or II (normal eye) and types III or IV (dry eye) is used to grade tear ferning patterns. Another grading scale is developed, which consists of five grades and is used in 0.1 increments. A grade > 2 is defined as dry eye. The tear ferning test has been applied to assess the quality of tears among birds, reptiles, and other animals, such as rabbits, cats, dogs, monkeys, horses, and camels. The tear ferning test has been used to investigate the effect of various disorders, such as the thyroid gland, diabetes, high body mass index, high blood cholesterol level, and vitamin deficiency on tear film. The effect of beverages such as green tea and peppermint that are rich in polyphenols on the tear film have been evaluated. Moreover, the effect of eyedrops on the tear film and addition of electrolytes on tear ferning patterns of eyedrops was studied. Conclusion: The tear ferning test is an easy-to-perform and inexpensive tool to evaluate the quality of tears in animals and human. It can be used in combination with other tests to detect dry eye symptoms. Keywords: Tear Ferning Test, Ferning Patterns, Tear Ferning Grading Scales, Tear Film Stability, Dry Eye
... Other studies have reported use of the TFT to determine the tear composition under healthy conditions and in patients with ocular abnormalities (9,10), in both humans (2) and other species (11)(12)(13). In humans, results have shown that the tear's osmotic density is the main trigger modifying the crystallization patterns (14); however, nothing is known for birds and reptile tears. ...
... Extracellular fluids, such as tears, maintain homeostasis through ionic balance and osmolarity (9,14). In human tears, the paracellular pathway of sodium secretion is supplemented by a transcellular pathway driven by apical sodium and potassium pumps in a primary fluid-secretion model in the lacrimal gland (6), and measurements by silicon-hydrogel (SiHG) contact lens showed sodium and chloride in high concentrations, similar to what was found in this study. ...
... This suggests that animals in contact with the aquatic environment have a specific tear composition aimed at maintenance and stability, including, for example, mucus. In human tears, thicker crystals result mainly from changes induced by the increased presence of mucus or macromolecules (1,11,14,32). ...
Article
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To compare tear electrolytes and tear crystallization patterns in birds and reptiles, tears were sampled by Schirmer tear test from 10 animals each of Ara ararauna, Amazona aestiva, Tyto alba, Rupornis magnirostris, Chelonoidis carbonaria, and Caiman latirostris, and 5 of Caretta caretta. The aliquots were pooled to assess concentrations of total protein, chloride, phosphorus, iron, sodium, potassium, calcium, and urea. For the tear ferning test, samples of each species were observed under a polarized light microscope at room temperature and humidity. Crystallization patterns were graded according Rolando and Masmali scales. There was more total protein and urea in owl and sea turtle tears, respectively, than in the other animals tested. Electrolyte balance was similar for all species, with higher sodium, chloride, and iron. In birds, Rolando-scale grades of tear crystallization patterns ranged from I to II, and from 0 to 2 using the Masmali scale; in reptiles, grades were II to IV (Rolando) and 2 to 4 (Masmali). Crystallization arrangements of some species had higher scores, as caimans and sea turtles, possibly due to different the tear composition. Marine and lacustrine species presented higher. The ionic balance of lacrimal fluids of birds and reptiles was similar to that in humans, with higher values of sodium and chloride. However, a similar tear composition did not influence the crystal morphology. Crystallization classification suggested that higher grades and types are due to the different microelements present in the tears of wild species.
... Silva et al. 20 investigated the tear ferning of healthy horses, and other researchers have applied TF tests to dogs, 21 capuchin monkeys, 22 and camels. 23 TF tests have not been described in mice, to the best of our knowledge, despite that mouse model being widely used for studies in ocular surface diseases. Hence, this study aimed to investigate the determining factors in tear ferning formation in healthy mice and to optimize testing conditions to establish a standardized protocol for further analyses under various ocular surface disease conditions. ...
... 13 Previous reports have described the application of TF tests in dogs, 21,28 horses, 20 and camels. 23 To the best of our knowledge, the use of TF tests in mice is being reported here for the first time. ...
Article
Full-text available
Purpose: Analysis of ferning formation after tear drop desiccation on a glass slide has been applied as a simple method to examine tear normality and is referred to as the tear ferning (TF) test. Despite use of the TF test in clinical settings and in some animals, thus far no TF test protocol has been developed for the mouse model. This study aimed to establish a mouse TF test protocol that can be used for dry eye research using the mouse as the study model. Methods: Tear sampleswere collected from 24 healthymice after repeated flusheswith 2, 5, 10, or 20 μL wash solutions, either 0.9% NaCl saline or sterile water, on the ocular surface. After sample collection, TF tests were performed at variable drop volumes (2– 20 μL), at a relative humidity of either 46% ± 2% or 53% ± 2%, and with temperature fixed at 24°C ± 2°C for comparison.Moreover, the influence of osmolarity (between 280 and 360 mOsm/L) and pH values (6.5–8.0) and the effect of centrifugation (4000 rpm, 10 minutes) on ferning formation were examined. Reproducibility and ferning storage stability were also determined. Results: An optimized protocolwas establishedwith relative humidity at 46%±2% and drop aliquot at 2 μL, using 0.9% NaCl saline as thewash solution.Using sterilizedwater as the wash solution did not result in any crystalloid formation. Centrifugation did not aid ferning formation in any of the samples. Higher osmolarity increased ferning formation fromgrades between 0 to 1 to grades between 2 to 3, but pH values that varied between 6.5 and 8.0 did not affect ferning formation. The establishedmouse TF test protocol also displayed reproducibility and storage stability. Conclusions: A TF test protocol for themouse modelwas established that could be used for comparative analyses under various ocular surface disease conditions. Translational Relevance: This mouse TF test protocol will facilitate the application of basic research into the mouse model to clinical care.
... Such scales can be converted to 0.1 increments, which make it easy to differentiate between various types of ferns. Both TF grading scales have been used to assess tear ferns in humans and animals (32)(33)(34)(35)(36)(37)(38). ...
Article
Full-text available
Objective: This study aimed to improve the tear ferning (TF) patterns in the sheep tears after the addition of various electrolyte solutions in different proportions. Animal Studied: Sheep were located at a small farm in the outskirts of Riyadh, Saudi Arabia. The sheep had no ocular disorders or diseases, and none of the female sheep were pregnant. Methods: Tear samples (20 μl) were collected from the right eyes of seven healthy sheep (five female sheep and two male sheep; age 7–36 months with an average of 17.0 ± 10.3 months). A tear sample (1 μl) from each sheep was dried on a microscopic glass slide at 22°C and <40% humidity. The TF patterns were graded based on the five-point grading scale in 0.1 increments. Homogenous mixtures were prepared by mixing tears from each sheep (0.5 μl) with various electrolyte solutions in different proportions (1:1, 1:2, 1:4, 1:6, 1:8, and 1:10). A sample of each mixture (1 μl) was dried on a glass slide, and the TF patterns for each mixture were observed, recorded, graded, and compared with those of the corresponding pure sheep tears. In addition, each sheep tear sample (0.5 μl) was diluted with pure water (0.5 μl) and the TF images were recorded and graded to test the dilution effect. Results: General improvement was noted in TF grades after the addition of electrolyte solutions, ranging from 1.7–1.4 to 1.3–0.3 regardless of the ratio between the electrolyte solutions and sheep tears within the mixture. TF grades of sheep tear samples improved significantly after adding different volumes of calcium chloride solution. Similar improvements in TF grades were observed when magnesium chloride hexahydrate and sodium dihydrogen phosphate solutions were used as the electrolytes. Some improvements in the TF grades occurred with the addition of potassium chloride to sheep tear samples. There was little improvement in TF grades after the addition of sodium chloride solution. Conclusion: Tear ferning grades of sheep tear samples improved when mixed with a number of electrolyte solutions at different volumes, in particular with calcium chloride or magnesium chloride solutions. Some improvements in TF grades were seen with sodium dihydrogen phosphate or potassium chloride solution added as the electrolyte. Clearly, divalent electrolytes lead to a greater improvement in TF grades of sheep tear samples as compared with sodium dihydrogen phosphate or monovalent electrolytes.
... The extension of the main arm is then interrupted in favor of side branches that bifurcate as more organic material accumulates at the ends [20]. Ferning patterns are complex and have proven themselves useful in the differentiation of dynamics and composition of tears collected from different animals [21]. In Figure 1, the four dry patterns clearly demonstrate the different dynamics occurring, due to the variation of components in the sample. ...
Article
Full-text available
Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.
... Type II, abundance of ferns with free spaces between them, type III has a rare or even unique fern, with large free spaces, and type IV demonstrates the absence of ferns, only some mucus threads being visible. (Figure 5) In the past, some authors considered that the appearance of fern leaf, the crystallized tear, is due to the amount of mucus present in the tear film (13). With the advent of new technologies, biochemical analyses identify other factors that are part of the TFT process, such as the concentration of ions in tears (14). ...
Article
Full-text available
Electromagnetic radiation (ER) emitted by mobile phones and other modern devices has potentially harmful effects on ocular tissue. Their effects on the eye surface and tear film are little known so far. The aim of this paper was to investigate the effects of ER emitted by the phone mobile on the tear film. For this study, we selected a total of 50 subjects, young, healthy, without chronic treatment, who are not contact lens wearers and who have no history of ophthalmic surgery. Schirmer I test, tear pH and tear ferning test (TFT) were performed on all subjects before (“-pre”) being exposed to ER emitted by the mobile phone and after (“-post”) exposure for 5 minutes, the pH and TFT of the tears were performed. Following the analysis of the obtained results, we found that there are significant changes in tear quality and increased tear pH, which over time can lead to tear film instability, damage to the eye surface and the appearance of dry eye syndrome.
... 9,24 In this controlled environment, there was a predominance of Rolando type I and Masmali grade 1 tear ferning patterns, similar to that described for healthy human subjects. 25,26 However, different results were observed in capuchin monkeys (Rolando II and III, Masmali 1 and 2), considered a peculiarity of the species. For healthy animals, Rolando types I and II and Masmali grades 0 and 1 crystallization patterns are related to normal and healthy conditions. ...
Article
SIGNIFICANCE The tear film promotes ocular surface health and protection through its various constituents' functions. The application of methods for ocular surface examination is essential in the research of diseases that affect the tear film. Rabbits have been used as a model to study some human ocular diseases and to test ophthalmic products. PURPOSE The aim of the present study was to determine the biochemical profile, osmolarity, and tear ferning patterns of rabbit tears. METHODS Ten rabbits (Oryctolagus cuniculus) were evaluated for tear osmolarity, tear ferning types and grades (using Rolando and Masmali scales), and biochemical analysis of total protein, urea, and electrolytes (chloride, phosphorus, iron, sodium, potassium, and calcium). RESULTS Median ± semi-interquartile range for tear osmolarity was 283.5 ± 7.5 mOsm/L. Tear ferning test grades were type 1.0 ± 0.5 on the Rolando scale and 1.0 ± 0.5 on the Masmali scale. Type I and grade 1 were the most commonly observed ferning classifications (60 and 50%, respectively) for the rabbits' tears. Results for electrolytes and other biochemical compounds were as follows: total protein 4.40 g/dL, urea 130.60 mg/dL, chloride 196.51 mEq/L, phosphate 7.35 mg/dL, iron 95.76 μg/dL, sodium 202.04 mmol/L, potassium 12.74 mmol/L, and calcium 11.53 mg/dL. CONCLUSIONS The results of the various tests described herein may serve as a basis for research using rabbits as an ophthalmic disease model and in the development of diagnostic and therapeutic agents used for ocular health.
Article
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Fern‐like crystallisation occurs in many body fluids when sampled and dried, including the tears. Tear ferning has been proposed as a diagnostic test of keratoconjunctivitis sicca (KCS), but the mechanism of reduced ferning in the dry eye is poorly understood. By microscopic examination of tear ferns and review of gynaecological and crystallographic literature, we seek an explanation for the reduced tear ferning in KCS. The physico‐chemical variables controlling the crystal ‘habit’ (viscosity, osmolality, protein content, and mucous quality or quantity) are examined to ascertain which might be responsible for the decreased ferning of dry eye tear samples. We hypothesise that a shift in the salt‐to‐macromolecule ratio, increased lipid contamination of mucus and altered tear rheology are the features of dry eye which best explain the altered ferning patterns in this condition.
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To describe the tear ferning test (TFT) in healthy horses and its correlation with other parameters for evaluating the ocular surface. Thirty male and female adult healthy horses (60 eyes), of no defined breed. Tear sample was collected with a microcapillary tube, placed on the surface of a glass slide, and allowed to dry at room temperature. The crystallization pattern was classified according to Rolando (Chibret International Journal Ophthamology, 1984; 2, 32). The program STEPanizer(©) stereology tool, version 1.0, was utilized for counting points on the digitally captured crystallization image. A conjunctival biopsy was performed. Tear ferning test was classified as Type I in 18 eyes (30%), Type II in 31 eyes (51.7%), and Type III in 11 eyes (18.3%), at a mean temperature of 27.3 ± 1.5 °C and relative humidity of 61.5 ± 5.7%. In the Type I crystallization, the count varied between 27 and 36 points (mean: 33.27 ± 2.40), in Type II between 22 and 31 points (25.42 ± 1.95), and in Type III between 13 and 25 points (16.82 ± 3.76). There was no statistical difference or correlation between the right and left eyes, nor was there a statistically significant influence (P < 0.05) on TFT by the factors evaluated. The mean goblet cells values were 50 ± 11.4 cells/field. All samples showed the presence of lymphocytes, plasmocytes, and eosinophils. Tear ferning test is easy to perform, without risks to the patient. Once standardized for horses, associated or not with the program STEPanizer(©) stereology tool, it is an additional method for evaluating the ocular surface. © 2015 American College of Veterinary Ophthalmologists.
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The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-omega-hydroxy fatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundances of various tear lipid amphiphiles reported herein are expected to have profound impact on the current understanding of the existent human tear film model.
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The present study was carried out to investigate the morphological and histomorphometrical characters of irides in dogs, camels, buffalos, and donkeys. The findings of the study revealed that, morphologically, the irides were consisted of an anterior border layer, a middle layer of connective tissue stroma and a posterior layer of pigmented epithelium. Interestingly, the anterior borders of all investigated animals were not enveloped by a distinct epithelial or endothelial lining, but on contrary, the posterior border was covered by pigmented epithelium. The constrictor and dilator iridial muscles were well developed in dogs, weakly developed in donkeys, and with an intermediate position in camels and buffalos. Morphometric analysis revealed significant species differences in the mean total thickness of the iris and its different layers. In addition significant differences were also found between the ratio of the means of different layers to the total thickness of the iris at the pupillary, middle and ciliary borders. In conclusion, these variations might be related to the different lifestyles and visual behaviour of the investigated animals.
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  The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs).   Camel corneas (of 6-, 8-, and 10-month-old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The 'AnalySIS LS Professional' program was used to analyze the collagen fibril diameter.   The camel cornea consists of four layers: the epithelium (227 μm), stroma (388 μm), Descemet's membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 μm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM.   The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.
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We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 µM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.
Article
A healthy tear film is very important for many major functions of the ocular surface. Dry eye disease is a significant clinical problem that needs to be solved but the poor correlation between clinical signs and reported symptoms makes it difficult for the clinician to apply a scientific basis to his clinical management. The problem is compounded by the difficulties of evaluating the tear film due to its transparency, small volume and complex composition. Practical insight into tear film composition would be very useful to the clinician for patient diagnosis and treatment but detailed analysis is restricted to expensive, laboratory-based systems. There is a pressing need for a simple test. The tear ferning test is a laboratory test but it has the potential to be applied in the clinic setting to investigate the tear film in a simple way. Drying a small sample of tear fluid onto a clean, glass microscope slide produces a characteristic crystallisation pattern, described as a 'tear fern'. This test is currently not widely used because of some limitations that need to be overcome but several studies have demonstrated its potential. Such limitations need to be resolved so that tear ferning could be used in the clinic setting to assess the tear film.
Article
Purpose: This paper reports on the development of a new tear ferning (TF) subjective grading scale, and compares it with the Rolando scale. Method: TF patterns obtained from tear film samples collected from normal and dry eye subjects in previous studies were collated into a large image library. From this library, 60 images were selected to represent the full range of possible TF patterns, and a further sub-set of 15 images was chosen for analysis. Twenty-five optometrists were asked to rank the images in increasing order between extreme anchors on a scale of TF patterns. Interim statistical analysis of this ranking found 7 homogeneous sub-sets, where the image rankings overlapped for a group of images. A representative image (typically the mean) from each group was then adopted as the grade standard. Using this new 7-point grading scale, 25 optometrists were asked to grade the entire 60 image library at two sessions: once using the 4-point Rolando scale and once using the new 7-point scale, applying 0.25 grade unit interpolation. Results: Statistical analysis found that for the larger image set, the Rolando scale produced 3 homogeneous sub-sets, and the 7-point scale produced 5 homogeneous sub-sets. With this refinement, a new 5-point TF scale (Grades 0-4) was obtained. Conclusions: The Rolando grading scale lacks discrimination between its Type I and II grades, reducing its reliability. The new 5-point grading scale is able to differentiate between TF patterns, and may provide additional support for the use of TF for both researcher and clinician.
Article
To investigate the tear proteome profiles of human, cow, sheep, and camel comparatively and to explore the difference of tear protein profiles among different species. Tears were collected from both eyes of 25 clinically healthy volunteers, 50 cows, 25 sheep, and 50 camels. Pooled tear protein samples were separated by SDS-PAGE and two-dimensional electrophoresis. Protein spots of differential expression were excised and subjected to in-gel digestion and identification by matrix assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrum analysis. Because of the incomplete genomic data of cow, sheep, and camel, a combined strategy of de novo sequencing and BLAST (Best Local Alignment Search Tool) homology searching was also used for protein identification. The differentially expressed proteins were validated by Western blot analysis. On comparison with human tears (182 ± 6 spots), 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in triphenylmethane dye-stained gels of cow, sheep, and camel tears, respectively. Similar high-abundant proteins (lactoferrin, lysozyme, etc.) were found in all tear fluids. Tear lipocalins have been identified in cow and sheep tears. BLAST searching revealed a 21-kDa protein, identical with human vitelline membrane outer layer protein 1 (VMO1) homolog, in camel tears. The Western blot confirmed that VMO1 homolog was present in both camel and sheep tears but not in human and cow tears. The comparative proteomic analyses of tears from healthy humans, cows, sheep, and camels were first reported. Differential protein expression existed in the tear among species, offering useful information for further study on tear proteins and the related ocular diseases.