Abstract
The purpose of this study was to establish normal values for tear production tests in different breeds of domestic rabbits. Healthy adult rabbits (n = 60; 120 eyes) of 2 different breeds (English angora and Dutch; n = 15 of each sex and breed) were used in this study. Tear production was measured by using the 1-min Schirmer tear test (STT), phenol red thread test (PRTT), and endodontic absorbent paper point tear test (EAPTT). In addition, horizontal palpebral fissure length was evaluated as a measure of ocular adnexal dimensions. Tear production (mean ± 1 SD) in English angora rabbits was 5.4 ± 1.6 mm/min according to the STT, 25.0 ± 2.7 mm in 15 s for the PRTT, and 18.8 ± 2.1 mm/min by the EAPTT; in Dutch rabbits, these values were 4.6 ± 1.2 mm/min, 23.6 ± 2.3 mm in 15 s, and 16.9 ± 1.7 mm/min, respectively. Only the EAPTT revealed a significant difference in tear production between English Angora and Dutch rabbits. These results provide reference values for tear production in English Angora and Dutch rabbits according to 3 different quantitative tear film assessment methods.
Abbreviations: EAPTT, endodontic absorbent paper tear test; HPFL, horizontal palpebral fissure length; PRTT, phenol red thread test; STT, Schirmer tear test
Domestic rabbits (Oryctolagus cuniculus) are popular pets and commonly referred to veterinarians for routine health care and solving health problems. Rabbits are also common laboratory research animals, because they are tractable and inexpensive to maintain. Ocular lesions of all segments of the eyes have been reported in wild, pet, and research animals.2 Rabbits have prominent, laterally placed eyes, allowing them a large field of vision, which is typical of prey species. They have an upper eyelid, a larger and thinner lower eyelid, and a third eyelid (or nictitating membrane). Rabbits normally blink infrequently, averaging 10 to 12 times hourly.12
The tear film is as vital to the normal function of the eyes as any anatomic component and is essential in the maintenance of corneal clarity.10,13 The aqueous layer is responsible for lubricating, oxygenating, and nourishing the cornea; flushing debris from the corneal surface; providing various antibacterial properties. The mucin layer allows the aqueous tear fluid to spread evenly over the cornea and allows the adherence of the tear film to the corneal surface.5 The lipid layer of tears contains waxy esters, sterols, triglycerides, cholesterol, and some polar lipids. This lipid mixture coats the surface of the tear film and retards tear evaporation.13
Quantitative tests for the evaluation of tear film include the Schirmer tear test (STT), phenol red tear test (PRTT), and endodontic absorbent paper point tear test (EAPTT). The STT is the most commonly used, standard method for tear film assessment. The results of various ophthalmic diagnostic tests have been reported for New Zealand white rabbits1,3,11 but not for any other breed. The purpose of the current study was to establish normal values for tear production in 2 breeds of domestic rabbits, English angora and Dutch, according to the STT, PRTT, and EAPTT.
Materials and Methods
Animals.
The study was approved by Iran Society for Prevention of Cruelty to Animals in accordance with Iranian ethical codes for studies on laboratory animals. The population consisted of 60 adult healthy rabbits (120 eyes) ranging in age from 1 to 2.5 y of 2 different breeds, English angora and Dutch; 15 male and 15 female rabbits from each breed were examined. All of the rabbits weighed between 1470 and 3680 g (mean ± 1 SD, 2514 ± 482 g) and were housed individually indoors beginning 7 d before the first test day in an air conditioned room at constant temperature (18 to 21 °C) and humidity (45% to 48%). The rabbits were exposed to a 12:12-h light:dark cycle, were fed a commercial rabbit diet, and had unrestricted access to water.
Study procedures.
The studied population was selected on the basis of normal physical and ophthalmic examination including CBC analysis, radiography, ophthalmoscopy (Binocular Indirect Ophthalmoscope, Welch Allyn, Rochester, NY), fluorescein staining (Fluorescein Glostrips, Nomax, St Louis, MO), slit lamp biomicroscopy (PSL Portable Slit Lamp, Reichert, Buffalo, NY), and tonometry (TonoVet, iCare, Tiolat, Helsinki, Finland).
Phenol red test threads (Zone-Quick, Menicon America, San Mateo, CA), endodontic absorbent paper points (Roeko Color, Roeko, Langenau, Germany), and Schirmer tear test strips (Opstrip, Ophtechnics, Haryana, India) from a single lot were used. A single examiner (SMR) performed all the ocular tests, examinations, and measurements. All tests were performed between 1400 and 1900 to minimize any possible variations associated with diurnal changes.
On day 1, PRTT was performed. The lower eyelid of each rabbit was everted, and the 3-mm folded head of the PRTT cotton thread was placed into the ventral conjunctival fornix for 15 s. The thread was then removed, and the portion of the thread that had become red in color was measured (in millimeters). On day 3, aqueous tear volume was measured using EAPPTT. One absorbent paper point was inserted in the lower conjunctival fornix of each eye; after 1 min, the paper point was removed, and its wetted portion was immediately measured by using a digital caliper graduated in millimeters. On day 5, an STT strip was placed in the inferior conjunctival fornix of one eye; after 30 s, another strip was placed in the contralateral eye. After 1 min, the strips were removed, and the amount of wetting was recorded (in millimeters per minute). In addition, the distance between the inner end of the caruncle and the inner end of the temporal canthus (that is, the horizontal palpebral fissure length [HPFL]) was measured by using a digital caliper.
Statistical analysis.
Statistical analysis was performed by using SPSS for Microsoft Windows (SPSS 20.0, IBM, Chicago, IL). Normality was tested by a one-sample Kolmogorov–Smirnov test. A paired-samples t test was used to compare PRTT, EAPTT, STT, and HPFL values obtained from the right and left eyes. Means and SD were calculated for left and right eyes individually and for all eyes combined. An independent-samples t test was used to compare mean PRTT, EAPTT, STT, and HPFL values according to sex and body weight in each group. Spearman correlation was used to evaluate the relationship between mean PRTT, EAPTT, STT, and HPFL values and body weight. A P value less than 0.05 was considered statistically significant.
Results
No rabbit showed any sign of ocular discomfort during the 24 h immediately after each tear production test. In addition, all rabbits were examined on day 17, and all were free of signs of conjunctivitis, keratitis, blepharitis, corneal ulceration, and intraocular diseases.
All of the continuous numeric data obtained from the population used in this investigation were normally distributed according to the one-sample Kolmogorov–Smirnov test (P > 0.2). Body weights (mean ± 1 SD) of English angora and Dutch rabbits were 2775 ± 461 g and 2254 ± 357 g (P = 0.01), respectively. For English angora rabbits, tear production was 5.4 ± 1.6 mm/min according to the STT, 25.0 ± 2.7 mm in 15 s by the PRTT, and 18.8 ± 2.1 mm/min in the EAPTT; for Dutch rabbits, these values were 4.6 ± 1.2 mm/min, 23.6 ± 2.3 mm in 15 s, and 16.9 ± 1.7 mm/min, respectively (Figure 1 and Table 1). For all tests, tear production did not differ between the left and right eyes or between male and female rabbits. Tear production according to EAPTT and HPFL differed significantly between English Angora and Dutch rabbits (P = 0.043 and 0.007, respectively); HPFL values were 16.0 ± 0.7 mm for English angora rabbits and 12.9 ± 2.8 mm for Dutch rabbits. There was no correlation between tear production values and the body weight or age of the rabbits. However the age and body weight were positively correlated (Pearson r = 0.708, P = 0.001; Spearman r = 0.638, P = 0.003), and both body weight (r = 0.637, P = 0.003) and HPFL (r = 0.669, P = 0.001) were positively correlated with the age of the rabbits. In addition, tear production values from the PRTT and STT were correlated (Pearson r = 0.543, P = 0.013; Spearman r = 0.511, P = 0.021) but not those from the PRTT and EAPTT or EAPTT and STT (Table 2).
Figure 1.
Box-and-whisker plots of tear production in the study population of 60 English angora and Dutch rabbits. STT, Schirmer tear test(mm/min); PRTT, phenol red thread test(mm/15s); EAPTT, endodontic absorbent paper tear test(mm/min).
Table 1.
Tear production in 60 rabbits, measured bilaterally
| Rabbits | STT (mm/min) |
PRTT (mm in 15 s) |
EAPTT (mm/min) |
|||
| Mean ± 1 SD | Range | Mean ± 1 SD | Range | Mean ± 1 SD | Range | |
| Male | 5.5 ± 1.3 | 3.5–7.5 | 25.3 ± 1.8 | 22.5–29.0 | 17.3 ± 2.4 | 14.5–22.0 |
| Female | 4.3 ± 1.5 | 2.5–7.0 | 23.0 ± 2.9 | 17.5–26.5 | 18.4 ± 1.5 | 15.0–21.0 |
| English angora | 5.4 ± 1.6 | 2.5–7.5 | 25.0 ± 2.7 | 24.0–29.0 | 18.8 ± 2.1a | 16.5–22.0 |
| Dutch | 4.6 ± 1.2 | 3.0–6.5 | 23.6 ± 2.3 | 17.5–26.0 | 16.9 ± 1.7a | 14.5–19.0 |
STT, Schirmer tear test; PRTT, Phenol red thread test; EAPTT, endodontic absorbent paper tear test
These values are significantly different (P = 0.043).
Table 2.
Correlation coefficients for bilateral phenol red thread tests (PRTT), Schirmer tear tests (STT), and endodontic absorbent paper tear tests (EAPTT) in 60 rabbits
| Correlation coefficient |
||
| Pearson | Spearman | |
| PRTT and STT | 0.54 | 0.51 |
| PRTT and EAPTT | 0.18 | 0.08 |
| STT and EAPTT | 0.15 | 0.19 |
Discussion
Rabbits have a large bilobed lacrimal gland (located ventrally behind the lower eyelid and adjacent to the lower orbital rim), Harderian gland (attached to the lower medial orbital wall), gland of the third eyelid, and venous sinus within each orbit.8 The lacrimal gland, Harderian gland, and gland of the third eyelid supply components of the precorneal tear film that are crucial to the health of the cornea.12 Rabbits are somewhat unique in having a single nasolacrimal punctum deep in the ventromedial conjunctival fornix. The nasolacrimal duct follows a tortuous route with several sudden constrictions, which are often sites of obstruction.4 Interbreed differences in the normal ophthalmic anatomy of rabbits have not yet been described. In the current study, all of the quantitative tear film assessment tests showed greater tear production in English angora rabbits than in Dutch rabbits. In addition, the HPFL was larger in English angora than Dutch rabbits. Although HPFL and blink frequency might directly affect the measurement of tear production and spread of tear film,9 whether this is the case has yet to be evaluated. In addition, fitting a conventional STT strip into a small palpebral fissure might be difficult and thus affect the measurement of tear production by this method. However, the rabbits in the current study had a mean HPFL of 12.98 mm, and it was easy to use standard STT strips.
For New Zealand white rabbits, normal tear production has been reported as 4.8 ± 2.9 to 5.3 ± 2.9 by STT,1,3,11 20.8 ± 3.7 by PRTT,10 and 13.8 ± 1.5 mm/min by EAPTT,11 respectively. For the STT, the tear production values of English angora and Dutch rabbits were close to the reference range for New Zealand White rabbits. However, tear production according to the PRTT and EAPTT was greater in both of the breed we tested than that reported for New Zealand White rabbits.
Although nasolacrimal outflow during STT measurement is reduced to zero, this situation does not compromise the determination of tear production. The STT strip absorbs all of the tears produced, and tear uptake by the test strip equals the tear production by the lacrimal and Harderian glands and gland of the third eyelid.14 In contrast, the argument might be made that PRTT and EAPTT measure tear volume in the conjunctival sac rather than assessing de novo tear production by lacrimal glands.
The parasympathetic nervous system provides the primary control of aqueous tear secretion. Basal secretion of tears is present continuously, and the increases in tear flow are stimulated by the nervous system.6 Although tear production is affected by environmental factors, time of measurement, and age of animals,7 we controlled for these factors in the current study. Future studies on the normal ocular anatomy of different breeds of rabbits are needed to help veterinarians interpret differences in normal physiologic processes, such as tear production, between different breeds of rabbits.
In conclusion, the current study used 3 quantitative tear -film assessment methods to determine reference values for tear production in English angora and Dutch rabbits.
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