Impact of stress on the tear production of healthy cats

Abstract

Objectives

This study aimed to determine the impact of acute stress on tear production in companion cats to provide a basis for minimizing stress-inducing stimuli during ophthalmic evaluations.

Methods

A total of 24 healthy owned cats (12 males, 12 females) of mixed breed, aged 8 months to 7 years, with no history of ocular diseases, were selected for the study. The cats were housed in individual cages under controlled conditions for 6 days. The Schirmer tear test-1 (STT-1) was performed in the morning (between 9:00 am and 11:00 am) using test strips from the same batch. The first test (without stress) was conducted on the fifth day of acclimation, and the second test (with stress) on the sixth day. The stress stimulus consisted of recordings of barking dogs, cats fighting and the murmuring of people. For both tests, the heart rate was assessed with a stethoscope before, during and after the tests, and the environmental stress level was also evaluated. Results are presented as mean ± SD and 95% confidence interval (CI).

Results

The study found that STT-1 values were significantly higher (P = 0.009) with stress (22.2 ± 6.0 mm/min [95% CI 19.9–24.6]) than without stress (17.5 ± 6.9 mm/min [95% CI 14.8–20.2]). Similarly, the heart rate was significantly higher (P = 0.028) in stress vs non-stress conditions (213.4 ± 37.5 beats per minute [bpm] [95% CI 198.7–228.1] vs 171.5 ± 28.6 bpm [95% CI 160.3–182.7], respectively), and the environmental stress score was significantly higher (P <0.001) in stress vs non-stress conditions (3.3 ± 0.5 [95% CI 3.1–3.5] vs 1.2 ± 0.4 [95% CI 1.1–1.4], respectively).

Conclusions and relevance

Stress increased tear production in cats. Although the mean STT-1 value obtained under stress conditions was within the normal range, stress can influence the test results. The use of cat friendly handling techniques facilitates execution of the STT-1.

Introduction

Stress can be defined as a condition or state in which an organism’s homeostasis is threatened by stressors. ¹ For cats, stressors can be loud or unfamiliar noises, sudden movements, unfamiliar places and objects, and the presence of strangers (humans, cats or other animals) entering their personal space. ² Stressors have additive effects, which means that the greater the number of stressors, the stronger the stress response. ³ Cats are susceptible to experiencing anxiety and stress from even minor changes in their routine or environment, including a simple visit to a veterinary clinic.^4,5 Ophthalmic examinations in cats can be challenging and short breaks may be needed during the eye examination to minimize stress. ⁶

The Schirmer tear test (STT) is commonly used in small animal ophthalmic examinations to quantify the aqueous component of tears.^7,8 Several factors can influence the test results, including ocular and systemic conditions, anatomical factors, aspects related to the STT strips, as well as physiological factors.^9,10 In cats, it was previously speculated that stress-induced sympathetic stimulation can temporarily reduce tear production, leading to lower STT values.^11,12 However, recent studies have debunked this speculation, establishing normative data for STT-1 in cats and showing that low STT-1 values are generally associated with clinical signs of dry eye syndrome.^{7,13 –18} In particular, a study that assessed tear production in cats under various environmental conditions showed that animals exposed to stressors, despite demonstrating a trend for increased STT-1 values, had results similar to those evaluated under stress-free conditions, demonstrating again that stress does not decrease STT-1 values in cats. ¹⁸

The aim of the present study was to consolidate previous findings regarding the impact of acute stress on tear production in healthy cats. We examined a different feline population under controlled settings (with an acclimation period) using advanced stress-reducing techniques, highlighting the importance of minimizing stress-inducing stimuli during ophthalmic examinations of cats in clinical practice.

Materials and methods

Animals

The study was conducted in March and April 2022 after approval from the local Committee on Ethics in the Use of Animals of the Universidade Federal da Fronteira Sul (UFFS) – Realeza. The owners of the cats agreed to their participation in the study and signed an informed consent form.

A total of 24 healthy cats (48 eyes) of mixed breed (12 males, 12 females), aged 8 months to 7 years and not neutered, were used in this study. The cats were all privately owned and needed to be docile, exhibiting no signs of aggressiveness during handling (ie, attempts to bite or scratch).

The inclusion criteria included the following: no history of ocular diseases; normal general physical examination (including heart and lung auscultation, pulse, rectal temperature, mucous membrane color, hydration status); normal ophthalmic examination (including STT-1, fluorescein 1% and lissamine green 1% staining, tear film break-up time, Jones test, slit-lamp biomicroscopy and direct ophthalmoscopy); normal complete blood count and biochemistry profile; and a negative result on a rapid immunochromatographic test for the detection of antibodies against feline immunodeficiency virus and feline leukemia virus antigens (FIV Ac/FeLV Ag Test Kit; Alere).

Cats with abnormalities in one or more of these tests were excluded from the study.

Acclimation and conditioning

Using a random number generator, the selected animals were randomly assigned to four groups, blocked only by sex, each containing six animals (three males and three females). One group per week was hosted at the University Veterinary Facility (UFFS – Realeza), allowing the cats to adapt to the environment and handling.

The animals were housed in individual stainless-steel cages measuring 240 × 60 × 60 cm, equipped with water and food (provided ad libitum), a litter box, a blanket and a hiding box. As environmental enrichment, each cage contained a toy suspended from the cage’s ceiling and two toys containing catnip (Nepeta cataria).

Each group was housed for 6 days in an environment with controlled noise, temperature (25°C) and lighting (12 h of artificial light and 12 h of darkness) facilitating their acclimation. Throughout their entire stay, a pheromone diffuser (FELIWAY Classic; Ceva Animal Health) was employed to provide a calming atmosphere.

To minimize the stress caused by the STT-1 itself, the animals were conditioned daily to the STT-1 under standardized conditions without additional stressors. For this purpose, 0.5 × 5 cm strips of common filter paper were placed in the central portion of the lower conjunctival sac for 1 min in both eyes. Immediately after the test, the animals were rewarded with cat-specific treats. Data were not collected during this conditioning period.

Notably, throughout the study period, the cats had contact with only two individuals; the examiner and the assistant.

Schirmer tear test-1

The STT-1 was conducted in the morning, between 9:00 am and 11:00 am, in a controlled environment. The test strips were placed in the central portion of the lower conjunctival sac for 1 min in both eyes. ⁶ The eyelids were not manipulated during the test. All test strips used belonged to the same batch (batch 13122132; DrogaVet). The order of testing (right or left eye) was randomized for each animal, and the strips were placed one immediately after the other.

During the examination, the animals were gently restrained on a table in a quadruped position with the assistance of a blanket. The heart rate (HR) of the cats was evaluated with a stethoscope immediately before the STT-1 (heart rate before [HRb]), during the test (10 s after the placement of the strips, heart rate during [HRd]) and after the test (30 s after the test, heart rate after [HRa]).

STT-1 without stress: This test was conducted on the fifth day of the acclimation period. The animals were individually carried, gently restrained with a blanket previously sprayed with pheromone spray (FELIWAY Classic; Ceva Animal Health), to a clinic room next to where the cats were staying. The room was equipped with a pheromone diffuser and specific cat music (Scooter Bere’s Aria by David Teie) was played at 60 decibels (measured using a sound level meter). Before starting the STT-1, a 5-min acclimation period was allowed. Throughout the procedure, cat friendly handling techniques were applied, following the guidelines proposed by Rodan et al. ¹⁹

STT-1 with stress: This test was conducted on the sixth day of the acclimation period. The animals were individually transported in a carrier to a room far from the housing, so the housed cats could not hear the auditory stressors. The room contained auditory stressors, such as recordings of barking dogs, cats fighting and the murmuring of people, played at 70 decibels (measured using a sound level meter). The recordings were played from the moment the cats entered the room until the end of the STT-1. In addition, another assistant unfamiliar to the cats was present; however, he did not interact with the cats. The measurement of tear production was performed 1 min after the cats arrived in the clinic room.

Environmental stress score

To assess the reaction of the cats to environmental stimuli on the days of STT-1 measurement, a descriptive numerical scale was applied to determine the environmental stress score before the start of the STT-1 (see Table 1 in the supplementary material). The cats were live-scored by the same two observers who also performed the STT-1. The assessment scale, adapted from the cat stress scoring system proposed by Kessler and Turner, ²⁰ categorized the animals using scores from 1 to 4 (relaxed, tense, fearful and terrified), with an additional 0.5 point added for those that did not fit entirely into one classification. The final score resulted from the consensus value between the two observers.

Statistical analysis

The normality of the samples was checked using the Shapiro–Wilk test, and the homogeneity of variances (homoscedasticity) was examined using Levene’s test. A P value of 0.05 was considered to indicate significant differences.

STT-1 values were obtained by averaging the measurements from both eyes of each cat after confirming with the Mann–Whitney test that there were no significant differences in tear production between the two eyes of each cat (without stress: P = 0.82; with stress: P = 0.89) or between males and females (without stress: P = 0.74; with stress: P = 0.43). Therefore, further results will be presented as the average of both eyes and for all cats regardless of sex (n = 24). The average HR was calculated as the mean of the HR before (HRb), during (HRd) and after (HRa) stress.

Mean ± SD and 95% confidence interval (CI) values were used to describe the variables. The Wilcoxon signed-rank test was performed to determine statistically significant differences between STT-1 and HR values with and without stress. The environmental stress scores with and without stress were compared using the t-test. To assess the correlation between environmental stress scores and HRs with STT-1 values, Kendall’s rank correlation coefficient (τ) was used.

All tests and graphics were performed using R software (R Foundation). The packages used included corrplot, ggplot2, stats, pacman, dplyr, rstatix and lmtest.

Results

Of the 25 cats evaluated, 24 met the inclusion criteria for participation in this study. The values obtained for STT-1 (mm/min), HR (beats per minute [bpm]) and environmental stress score were tabulated for each animal.

When grouped by the mean (left and right eyes combined), the STT-1 values were significantly higher (P = 0.009) during stress-inducing stimuli (22.2 ± 6.0 mm/min [95% CI 19.9–24.6]) than during stress-free examinations (17.5 ± 6.9 mm/min [95% CI 14.8–20.2]) (Figure 1). In addition, 23/24 animals showed an increase in tear production on the day with stress stimuli.

Figure 1

Box plot for the variable Schirmer tear test-1 (STT-1), measured in mm/min, with median values, 1st and 3rd quartiles, and lower and upper limits for tests with and without stress. The two groups were significantly different (P = 0.009). Individual points represent outliers; X represents a mean value. Sample size = 24

The same pattern was observed for the HR and environmental stress score. The mean value for HR was 171.5 ± 28.6 bpm (95% CI 160.3–182.7) without stress and 213.4 ± 37.5 bpm (95% CI 198.7–228.1) with stress (P = 0.028) (Figure 2). The mean value for the environmental stress score without stress was 1.2 ± 0.4 (95% CI 1.1–1.4) and with stress 3.3 ± 0.5 (95% CI 3.1–3.5; P <0.001) (Figure 3).

Figure 2

Box plot for the variable heart rate (HR), measured in beats per minute (bpm), with median values, 1st and 3rd quartiles, and lower and upper limits for tests with and without stress. The two groups were significantly different (P = 0.028). Individual points represent outliers; X represents a mean value. Sample size = 24

Figure 3

Box plot for the variable environmental stress score, classifying the animals from 1 to 4, with median values, 1st and 3rd quartiles, and lower and upper limits for tests with and without stress. The two groups were significantly different (P <0.001). Individual points represent outliers; X represents a mean value. Sample size = 24

The STT-1 value was neither significantly correlated with the environmental stress score (P = 0.091) nor with the HR (P = 0.065). Moreover, the environmental stress score was not significantly correlated with the HR (P = 0.663).

Discussion

The long-standing belief that sympathetic stimulation induced by stress can temporarily reduce tear production in feline species^11,12 has been contradicted by recent evidence.^{7,13 –18} Likewise, the findings of the present study do not support this theory because the STT-1 values were significantly higher during the stress-inducing intervention than during the day without stress stimuli. Notably, 23/24 evaluated animals showed an increase in tear production during the stress-inducing intervention.

In their evaluation of tear production in cats in various environmental settings, Sebbag et al ¹⁸ also found that stress does not decrease tear production. In fact, the cats under stress conditions showed a trend for an increase in lacrimal production; however, these authors did not find significant differences in STT-1 values between animals stimulated with stressors and those without.

Another study conducted by Di Pietro et al ¹⁷ with the aim of assessing how hospitalization (considered a stressful environment) disrupts the daily rhythm of tear production in cats found no difference in STT-1 values between animals evaluated in comfortable housing conditions and those that were hospitalized.

In contrast with Sebbag et al ¹⁸ and Di Pietro et al, ¹⁷ we found a significant difference in STT-1 values between cats with and without exposure to stress stimuli. This may be explained by the fact that the acclimation period and daily conditioning of the animals, combined with cat friendly handling, likely mitigated the stress caused by the test itself, allowing a more accurate assessment of the influence of acute stressors on tear production. This study highlights the importance of employing stress-reducing strategies when evaluating tear production in feline patients.

Although the STT-1 values obtained during the stress-inducing intervention were significantly higher than those under stress-free conditions, our results for both groups were consistent with the reference range of the STT-1 previously reported for cats.^7,13,18

Despite sex differences being reported in a retrospective study of dry eye (keratoconjunctivitis sicca) in dogs, ²¹ most research findings indicate that tear production is equivalent in both sexes of healthy dogs^22,23 and cats.^7,13,16 The neuter status has been reported to influence the STT-1, showing higher values in neutered cats.^13,16 In the context of our study, no significant difference in STT-1 values was observed between male and female cats. These findings support the idea that sex is not a factor significantly influencing tear production in feline patients. However, we chose to enroll only intact cats to minimize possible variations caused by the neuter status.

Some cats may experience discomfort during the STT.^9,15 It was observed that when using a cat friendly handling approach on the day without stress-inducing intervention, the animals tolerated the positioning of the test strips well, requiring minimal restraint. However, during the stress-inducing intervention, many of the cats attempted to escape from the restraint and to remove the test strips.

The activation of the sympathetic nervous system in response to acute stress results in increased HR and systemic blood pressure, pupil dilation (mydriasis), elevated blood glucose and free fatty acid levels, as well as a state of excitement. ²⁴ During the stress-inducing intervention in this study, an increase in HR was observed, indicating that the experimental model used to induce stress was effective. In addition, higher environmental stress scores were also noted during the intervention.

The exact mechanism by which sympathetic innervation contributes to tear secretion is still unknown. However, sympathetic nerves play a role in stimulating tear secretion by releasing norepinephrine and neuropeptide Y. ²⁵ Whitwell ²⁶ demonstrated that the STT value in a cat was higher when sympathetic innervation of the lacrimal gland was electrically stimulated compared with the unstimulated contralateral eye. It is presumed that sympathetic activation during the stress response stimulates tear secretion, but further studies are necessary to fully elucidate this process.

In the present study, no correlation was found between HR and STT-1 values. While the exact mechanism of tear secretion remains not fully elucidated, it is presumed that the HR does not directly influence tear production. We also did not find correlations between the environmental stress score and STT-1 values. One possible explanation for this lack of correlation is that the stress score proposed by Kessler and Turner, ²⁰ although widely used, may lack specificity in evaluating and quantifying stress.^27,28

The present study has some limitations. The small sample size and heterogeneity of the group are limiting factors, and the differences observed may not necessarily be representative of the entire population; however, our study design aimed to reduce potential variations caused by breed, age, sex and animal behavior. In addition, in this study, all test strips belonged to the same batch, and the tests were conducted during the same time of day, performed by the same examiner, under controlled conditions, to minimize potential influences on the results. We also recognize that the performance of the STT-1 on two different days is a relevant limitation, as well as the non-randomization of the test order (without stress and with stress). Since we aimed to conduct the tests at the same time of day to minimize circadian rhythm-related variations, ¹⁰ the tests were conducted on two different days for logistical reasons. Regarding the non-randomization of the test order, we chose to perform the stress-free test first to avoid negative memory association with the execution of the STT-1 with stress, as cats can exhibit episodic-like memory.^29,30

Conclusions

The findings of this study suggest that acute stress may increase tear production in cats; however, the STT-1 values obtained during the stress-inducing intervention were within the normal range. In addition, it was observed that the use of cat friendly handling techniques facilitated the examination, keeping the animals calmer during the procedure. We encourage performing the STT-1 in cats in a stress-free environment, not only to mitigate the influence of stress-related variations but also for the comfort of the cat.

By understanding how acute stress affects tear production in cats, we can implement stress-reduction strategies during eye examinations. This knowledge is crucial for improving tear assessment in feline patients and ultimately their overall eye healthcare.

Supplemental Material

Table 1:

Environmental stress score