Abstract
Objective—To determine baseline tear pH in dogs, horses, and cattle by use of a microelectrode.
Animals—28 dogs, 24 horses, and 29 cattle.
Procedures—Under manual restraint, tears were collected from each subject's left eye with cotton spears. A Schirmer tear test was performed in the right eye. Tears were extracted from the spears by centrifugation. Tear volume was measured, pH was determined with a microelectrode, and total solids (TS) concentration was measured by refractometry.
Results—Mean ± SD pH of tears in cattle, dogs, and horses was 8.32 ± 0.14, 8.05 ± 0.26, and 7.84 ± 0.30, respectively. Tear pH was significantly higher in cattle versus dogs and horses and in dogs versus horses. Mean ± SD TS concentration in horses, cattle, and dogs was 2.04 ± 1.29 g/dL, 1.07 ± 0.60 g/dL, and 0.33 ± 0.18 g/dL, respectively. Total solids concentration was significantly higher in horses versus cattle and dogs and in cattle versus dogs. Schirmer tear test results for all animals were within the species reference range.
Conclusions and Clinical Relevance—Tear pH in all 3 species differed from that of published blood pH values and the pH of common topically administered ophthalmic medications. These fndings may have implications for variations in ocular flora and defense mechanisms, susceptibility to ocular disease, and success or comfort of topical treatment.
Tear pH baseline values are reported for humans,1–5 rabbits,5 mice,6 llamas,7 and cattle,7 and preliminary results are reported for horsesa (Table 1). The pH of the tears of humans and other animals has been measured with various methods including pH meters,8 glass electrodes,1 fluorescence microscopy,6 fluorescent probes,5 and more; the use of different methodologies probably contributes to the wide range of reference values reported in humans.
Mean ± SD and range values obtained from the literature for tear pH in humans and other species.
| Species | Technique | Range | Mean ± SD | Reference |
|---|---|---|---|---|
| Humans | pH indicator paper | 7.0–8.5 | 7.5 | Adler et al3 |
| pH meter | 6.92–8.00 | 7.45 ± 0.16 | Carney and Hill2 | |
| pH microelectrode | 6.5–7.5 | 6.93 ± 0.27 | Norn4 | |
| Fluorescent probe | NA | 7.83 ± 0.2 | Chen and Maurice5 | |
| Rabbits | Fluorescent probe | 7.9–8.5 | 8.2 | Chen and Maurice5 |
| Mice | Fluorescence microscopy | NA | 7.59 ± 0.2 | Ruiz-Ederra et al6 |
| Horses | pH indicator paper | 8–8.6 | 8.33 ± 0.15 | Lowe and Crispina |
| Llamas | pH meter | NA | 8.05 ± 0.01* | Gionfriddo et al7 |
| Cattle | pH meter | NA | 8.10 ± 0.01* | Gionfriddo et al7 |
The SD values for these data represent the inherent SD of the pH meter used for a single pooled sample of tears.
NA = Not applicable.
The importance of pH as a protective barrier and its various values are described both in veterinary and human medicine for organs such as the skin,9,10 stomach,11 and vagina.12 In these organs, changes in pH are associated with atopic dermatitis,13 gastritis,14 and vaginal infections,15 respectively. In humans, tear alkalization is associated with various ocular diseases such as nasolacrimal duct stenosis, keratitis, and especially mycotic keratitis.4 Nonetheless, the investigators of the study reported here could not find data clarifying the role of pH as a protective barrier in the eye and found little data regarding its baseline values and variations in domestic animals (Table 1).
pH can influence the ocular milieu in various ways and may be influenced by it. In humans, the tear protein lipocalin has pH-dependent ligand binding properties; changes in pH induce conformational changes in tear lipocalin, which affect its ability to bind lipids.16 Moreover, pH contributes to permeability barrier formation and antimicrobial defenses in the skin10 and is likely to have a similar effect in the eye, potentially influencing its flora and defense mechanisms. For example, in humans with untreated ocular rosacea, tear pH is significantly more alkaline than in healthy individuals or those with other ocular diseases, indicating a relationship between the disease and tear pH.17 Ocular diseases do not only affect tear pH; tear electrolytes and proteins also change in ophthalmic diseases, such as in the formation of dacryoliths.8
Determining baseline tear pH is important in choosing appropriate ophthalmic medication for reasons of both comfort and drug stability in various species. Most topically administered ophthalmic preparations used in veterinary medicine are formulated for primary use in humans. The pH of common ophthalmic preparations varies greatly, from 4.5 to 11.5.18 The closer the ophthalmic solution's pH is to tear pH, the less irritating the solution is to the eye.19,20 The pH comfort zone for topically administered ophthalmic medication in humans is quite narrow, ranging from 6.5 to 7.8,21 with a recommendation that drugs be instilled with a mean ± SD pH of 7.2 ± 0.2.19 Although such alkaline pH values result in drugs that may be less irritating, these formulations are less stable and thus have a shorter shelf life.22 For example, both pilocarpine and physostigmine are non-irritating to the eye and active at a pH of 6.8, but are subject to a substantial loss of chemical stability within 1 year, as opposed to drugs with a pH of 5, which are stable for years.22 Furthermore, it is quite possible that the local irritation associated with the use of some topically administered drugs such as highly concentrated pilocarpine solution may be attributable to pH values that are outside the comfort zone.
Determining baseline values of tear film pH in various animal species is essential to future investigation of its influence on ocular surface health and patient comfort in veterinary medicine. The purpose of the study reported here was, therefore, to determine tear pH values in dogs, horses, and cattle by use of the same methodology in each species, while attempting to control for seasonal, diurnal, and geographic effects by performing all measurements in a 2-week period, at the same time of the day, and within a small geographic region.
Materials and Methods
Study population—Twenty-eight dogs, 24 horses, and 29 cows were studied. Subjects were free of systemic disease and did not receive any medical treatment at the time of the study. The adult canine population of the Israel Guide Dog Center for the Blind was selected in its entirety. Of the 28 dogs, 4 were sexually intact females, 12 were spayed females, and 12 were neutered males. The mean ± SD age of the canine population was 25.0 ± 12.7 months. Canine breeds included 15 Labrador Retrievers, 11 Golden Retriever–Labrador Retriever crossbreds, 1 Golden Retriever, and 1 German Shepherd Dog. Twenty-nine Holstein-Friesian dairy cows with a mean ± SD age of 39.5 ± 12.7 months were selected from a single herd at the Volcani Center of the Agricultural Research Organization. Twenty-four horses of which 6 were females and 18 were geldings, with a mean ± SD age of 163.1 ± 62.2 months, were selected from the Therapeutic Riding Center, Ramat-Gan. Equine breeds included 12 grade horses, 5 grade ponies, 2 Thoroughbreds, 2 Tinkers, 1 Haflinger, 1 Miniature horse, and 1 Welsh pony. All experimental procedures were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Institutional Animal Care and Use Committee. Institutional consent was provided for the examination of all animals.
Sample collection—Animals were examined during 2 weeks in April 2013 between 6:30 am and 12:30 pm. Cattle and horses were each examined over 2 days and all dogs were examined on the same day. The external temperature was 18° to 20°C during the study, excluding the second day of equine examination when the temperature was 24° to 26°C. Dogs were examined in an examination room, and horses and cattle were examined in a shaded roofed area. Fractious animals were excluded from the study. All subjects were accustomed to frequent handling, thus allowing use of manual restraint alone. No topical anesthetic or other solutions were applied prior to tear collection.
Tears were collected from the left eye of each subject with a sterile cotton surgical spear.b A spear was placed in the medial aspect of the lower conjunctival fornix and was left 1 minute for horses and cattle and 2 minutes for dogs (Figure 1). After removal of the spear from the eye, its plastic handle was trimmed and the spear was placed with the sponge directed upward in a 1.5-mL Eppendorf test tube. An STTc was performed in the right eye. The STT strip was maintained in the lateral aspect of the lower conjunctival fornix for 30 seconds in horses and cattle and for 1 minute in dogs. The eye in which the examination commenced was chosen randomly. An examination of the anterior ocular segment and adnexa was then performed with direct illuminationd and magnification loupes.e The examination concluded with fluorescein staining of both corneas.f Animals with abnormalities of the anterior ocular segment or adnexa were excluded from the study.
Photograph of a horse with a cotton spear placed in the medial aspect of the lower conjunctival fornix to collect tears. Notice the open eye and apparent lack of discomfort during sampling.
Citation: American Journal of Veterinary Research 75, 5; 10.2460/ajvr.75.5.494
The test tubes were transported to a laboratory located < 10 miles from the area of sampling and centrifugedg at 0.36 × g for 5 minutes. A digital pH meterh with a range of 0 to 14 was used to determine tear pH by use of a glass pH microelectrode.i The pH meter was calibrated according to the manufacturer's instructions to an accuracy of ± 0.01 pH units. After centrifugation, the spear was removed from the test tube and the glass microelectrode was placed in the collected tear fluid in the tube in a manner that covered its tip. A pH measurement was discarded for 1 cow sample and 4 horse samples that did not have sufficient tear fluid to cover the microelectrode tip. pH measurements were recorded, and a sample of tear fluid was applied for the measurement of TS concentration with a clinical handheld TS refractometer. Tear volume was measured by approximation according to volume markings on the test tube and a model test tube with manually added markings at 0.01-mL intervals, measured by fluid dispensed from a 1-mL tuberculin syringe. pH measurement was repeated 24 hours later for the 14 cow samples measured on the first day to examine pH changes over time; in the interim, the test tubes were maintained sealed and at room temperature (25°C). All measurements were made at room temperature.
Statistical analysis—Statistical analysis was performed with commercial software.j All variables analyzed in this study were quantitative, excluding sex and reproductive status; descriptive statistics were calculated for these data. The Pearson correlation coefficient (r) was calculated to evaluate the relationship between various quantitative values. The paired Student t test was used to compare the pH measured in samples from 14 cattle on the first trial day and remeasured in the same samples 24 hours later. The independent-samples t test was used to compare the pH measured in samples from horses examined on the first and second days at different ambient temperatures. This test was also used to compare STT values and tear volume between horses and cattle as well as pH, STT values, and tear volume between females and males for horses and dogs. Oneway ANOVA was applied and followed by the Dunnett T3 post hoc test to examine the differences between species for pH, TS concentration, and tear volume. No adjustment was made for multiplicity because the other nonprimary variables were exploratory. All tests applied were 2-tailed, and P ≤ 0.05 was considered significant.
Results
Descriptive statistics for STT values, pH, TS concentration, and tear volume in dogs, horses, and cattle were determined (Table 2). Differences were observed among all species, with pH significantly higher in cattle versus dogs and horses, and in dogs versus horses (Figure 2). No difference was found between pH measured in 14 cattle on the first trial day and pH remeasured in the same samples 24 hours later (P = 0.92). No difference was found between pH measured in horses on the first day versus the second day of measurement (P = 0.36).
Box-and-whisker plots of tear pH in cattle (n = 28), dogs (28), and horses (20). For each species, the box represents the 25th to 75th percentile, the line within the box represents the median, and the whiskers represent the range. Outliers are not represented.
Citation: American Journal of Veterinary Research 75, 5; 10.2460/ajvr.75.5.494
Descriptive statistics for variables measured in tears of cattle, dogs, and horses.
| Species | Statistic | STT* | pH | TS (g/dL) | Volume |
|---|---|---|---|---|---|
| Cattle | Mean ± SD | 25.52 ± 6.10 | 8.32 ± 0.14 | 1.07 ± 0.60 | 0.12 ± 0.05 |
| Median (range) | 27.00 (12.00– 35.00) | 8.31 (8.13–8.80) | 0.90 (0.30–3.20) | 0.10 (0.03–0.23) | |
| No. of animals | 29 | 28 | 29 | 29 | |
| Dogs | Mean ± SD | 23.96 ± 4.25 | 8.05 ± 0.26 | 0.33 ± 0.18 | 0.15 ± 0.05 |
| Median (range) | 23.50 (13.00– 34.00) | 8.04 (7.37–8.62) | 0.30 (0.00–0.070) | 0.15 (0.08–0.30) | |
| No. of animals | 28 | 28 | 28 | 28 | |
| Horses | Mean ± SD | 22.25 ± 5.81 | 7.84 ± 0.30 | 2.04 ± 1.29 | 0.10 ± 0.07 |
| Median (range) | 23.00 (12.00– 35.00) | 7.86 (7.13–8.35) | 1.60 (0.50–6.10) | 0.10 (0.02–0.25) | |
| No. of animals | 24 | 20 | 24 | 24 |
For cattle and horses, the STT units are mm/30 s; for dogs, the STT units are mm/min.
The correlation between TS concentration and tear volume across all species was r = −0.55 (P = 0.01), and it was not improved when examined for each species separately. The TS concentration was significantly higher in horses versus cattle and dogs, and in cattle versus dogs (Figure 3). A significant difference was found between horses and cattle for STT values (P = 0.03), but not tear volume (P = 0.26), with STT value slightly higher in cattle; we did not compare STT values and tear volume of cattle and horses to that of dogs, because tear collection and STT measurement times were longer in dogs. The correlation found between STT value and tear volume across all species was r = 0.22 (P = 0.01), and it was not improved when examined for each species separately.
Box-and-whisker plots of tear TS concentrations in cattle (n = 29), dogs (28), and horses (24). Outliers are not represented
Citation: American Journal of Veterinary Research 75, 5; 10.2460/ajvr.75.5.494
No significant difference was found between males and females in either horses or dogs for pH, TS, STT values, and tear volume. Little correlation was found between age and pH, STT value, and TS across all species (r ≤ 0.35; P = 0.01). Correlations were not improved when examined for each species separately. The correlations found across all species between pH and TS, STT value, and tear volume were r = −0.37, r = 0.34, and r = 0.20, respectively (P = 0.01). These correlations were not improved when examined for each species separately.
Most subjects had little to no signs of discomfort during the examination and tear collection. For the 3 species in this study, a sufficient tear volume with a mean of 100 μL (Table 2) was collected easily and rapidly, allowing for reliable measurements.
Discussion
The tear pH of the 3 species was more alkaline than most values reported in humans, so it is likely that the ocular pH comfort zone for these species is more alkaline than in humans. Because veterinarians often prescribe topically administered ophthalmic preparations formulated for humans, one could assume that production of more alkaline ophthalmic drug preparations could increase the ocular comfort of treated animals. However, alkalization of these preparations would decrease their shelf life20 and would come at a substantial cost or require other modifications, which are not always possible, for improved drug stability.
Tear pH varied significantly among the 3 species. The study population included only systemically and ophthalmologically healthy animals. The mean STT values were within the reference ranges reported in the literature for each species (mean ± SD, 18.64 ± 4.47 mm/min to 23.90 ± 5.12 mm/min for dogs; 15 to 20 mm/30 s for horses; and 24.18 ± 6.5 mm/30 s for cattle).23 The study population was homogeneous for cattle; relatively homogeneous for dogs, which included only mesaticephalic breeds of which most were retrievers; and highly heterogeneous for horses (mostly grade horses). Breed differences may have accounted for the greater variability of pH and TS concentrations detected in horses, compared with dogs and cattle, and conversely, the lack of breed variability in the cattle could have caused a selection bias. Any possible association between skull conformation or breed and tear pH values in dogs and cattle should be investigated in future studies. As in humans, tear pH was not associated with sex and was only weakly correlated with age.4
The mean tear pH measured in horses, 7.84, was lower than the 8.33 value reported in a preliminary study by Lowe and Crispin.a The mean tear pH measured in cattle, 8.32, was higher than the 8.10 value measured in a pooled sample of cow tears by Gionfriddo et al.7 These differences could be attributed to the use of different techniques (ie, pH paper used in horses and tear pooling used in cattle in previous studies versus use of a glass microelectrode and individual animal measurements in the present study) or to diurnal variations, different horse breeds, different geographic locations, or other factors. The study performed in horses by Lowe and Crispina was a preliminary study published as an abstract; therefore, we could not find published data that would allow comparison with our controlled variables. The study performed in cattle by Gionfriddo et al7 used a single pooled sample for all subjects, so the mean could have been influenced by extreme measurements; therefore, although the bovine population sampled was of the same breed and sex, it is difficult to draw conclusions from this comparison. Indeed, tear pH of healthy humans measured with different techniques varies greatly, ranging from 5.2 to 8.6, and has diurnal variations, with tears markedly more alkaline in waking hours.2 We accounted for all these variables, except horse breed, by determining pH with the same methodology for all 3 species within a 2 week period and by testing animals in 1 geographic region at the same time of day, with animals subject to similar husbandry.
Although we did not measure blood pH, the tear pH was clearly more alkaline than arterial pH in dogs (7.36),24 horses (7.41),25 and cattle (7.45).25 Tear electrolyte composition also differs from blood electrolyte composition in dogs and horses.k The differences between tear pH and the physiologic pH of other bodily fluids may have clinical implications. Although serum pH is expected to be in the ocular comfort zone of dogs, horses, and cattle, it is likely that continuously exposing the eye to fluids that are more alkaline or acidic than tears could cause ophthalmic discomfort and irritation. Parotid duct transposition for the treatment of keratoconjunctivitis sicca is known to be associated with complications, often including discomfort and saliva intolerance.26 The mean ± SD salivary pH of dogs is 8.53 ± 0.34,27 which is more alkaline than canine tears and could possibly contribute to irritation following parotid duct transposition.
There was a significant difference in tear TS concentrations between dogs, cattle, and horses. Although TS concentrations in a fluid are expected to be different and slightly higher than TP concentrations, when comparing our results to the literature, the low mean tear TS concentration we measured in dogs (0.33 g/dL) was similar to the mean tear TP concentration reported in humans (0.25 g/dL),8 but lower than the mean ± SD tear TP concentration reported in dogs (0.63 ± 0.04 g/dL)28 and cats (0.58 g/dL).29 The tear TS concentration measured in cattle (1.07 g/dL) was similar to the TP concentration determined previously with a bicinchoninic acid assay protocol (0.97 g/dL).7 The precorneal tear film contains soluble proteins that vary among species, including antibodies, matrix metalloproteinases, and others, which all contribute to the defense mechanisms of the cornea.7,30 In camels, a difference was detected in the protein profile of tears between winter and summer,31 and it is possible that these variations exist in other species.
The sampling technique we used was easy to perform, and animals appeared comfortable during examination and sampling, necessitating no sedation and only gentle manual restraint and handling. Nonetheless, it is difficult to determine how the sampling technique may influence reflexive tearing, tear composition, and perhaps tear pH, volume, and TS concentration. In a preliminary study, we attempted tear collection in cats, but tear volume was insufficient to obtain a reliable pH measurement. Results of the study reported here indicated that there was a significant difference in tear pH and TS concentration among cows, dogs, and horses. Although the tear collection technique used in this study may be unsuitable for small species or species with low tear production values, it was easy to perform and caused little discomfort in the species we examined. Further studies are warranted to determine the effect of tear pH on ocular comfort during topical ophthalmic treatment and on the efficacy of such treatments. Tear pH in animals during ocular disease also warrants further investigation.
ABBREVIATIONS
| STT | Schirmer tear test |
| TP | Total protein |
| TS | Total solids |
Lowe CR, Crispin SM. Normal equine tear pH as measured with pH paper (abstr). Vet Ophthalmal 2003;6:348.
Eye Spear 9001, Hurricane Medical, Brandenton, Fla.
TearFlo, HUB Pharmaceuticals LLC, Rancho Cucamonga, Calif.
3.5-V Finnoff ocular transilluminator, Welch Allyn Inc, Skaneateles, NY.
HR 2.5× high resolution binocular loupes, Heine, Herrsching, Germany.
BioGlo, HUB Pharmaceuticals LLC, Rancho Cucamonga, Calif.
Mikro 20, Hettich Zentrifugen, Tuttlingen, Germany.
PL-600, MRC Ltd, Holon, Israel.
CR-5028, MRC Ltd, Holon, Israel.
SPSS for Windows, version 18.0, SPSS Inc, Chicago, Ill.
Taylor L, Ben-Shlomo G. Biochemical composition of precorneal tear film in normal dogs and horses (abstr). 43rd Annu Conf Am Coll Vet Ophthalmol 2013;16:1–21.
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