Tear Ferning Test in Sheep: Scale-Based Analysis, STEPanizer Method, and Correlation With Conjunctival Histological Findings

To evaluate the precorneal tear film (PTF) of clinically healthy sheep using the tear ferning test (TFT) and investigate its association with conjunctival histological parameters.

One hundred and thirty-four eyes from 67 adult sheep.

Tear samples were collected and graded by three evaluators using the Rolando and Masmali grading scales. Digital stereological analysis of tear ferning patterns was performed using STEPanizer to quantify crystalline points. Conjunctival histology included assessment of inflammatory cell infiltration and goblet cell (GC) density. Data were analyzed using descriptive statistics, Spearman's correlation, Kendall's coefficient of concordance, and simple linear regression.

Rolando and Masmali grading scales showed strong positive correlations between right and left eyes (r = 0.922 and 0.886, respectively; p < 0.0001), demonstrating high consistency between eyes. STEPanizer crystal point counts were strongly and negatively correlated with TFT grades on both scales (r = -0.826 to -0.890; p < 0.0001). Linear regression showed that stereological measurements explained 66%-83% of the variation in TFT grades (R² = 0.66-0.83). Interobserver agreement was acceptable (Kendall's W = 0.69; p < 0.05). No significant correlations were found between TFT grades and histological parameters. Mild lymphocytic infiltration predominated (83.96%), while only weak negative associations were observed between TFT grades, GC density, and inflammatory infiltrate.

In clinically healthy sheep, TFT patterns appear to be influenced primarily by tear film physicochemical characteristics rather than routine conjunctival histological findings. Combining TFT with digital stereological analysis provides an objective and feasible approach for PTF characterization in veterinary ophthalmology.