Analysis of sperm morphology is a fundamental test for the examination of sperm quality and the breeding potential of stallions [[1], [2], [3]]. Several techniques have been used to evaluate stallion sperm morphology, including background stains such as eosin-nigrosin (EN) [[4], [5], [6]] or Romanowsky-stain variants [[7], [8], [9]] evaluated by light microscopy; or wet mounts of fixed samples analyzed by phase-contrast (PH) [1,5,7] or differential interference contrast (DIC) microscopy [1,3,9,10].

While the technique and evaluator may play a role in the determination of the actual values from a sperm sample, the statistical analysis performed to evaluate differences among techniques may also influence the interpretation of the results. In addition, other variables that influence the outcome include operator training and experience and the sperm quality (high vs. low percent of normal sperm) of the sample evaluated [5,6,11,12]. For instance, the ability of the operator to identify a sperm abnormality may vary based on the technique which can include both the actual preparation of the sample (fixed vs. stain) and the magnification and microscope type (light vs. PH [11,12]).

Technique validation should include a variety of sperm quality levels to determine the ability of the technique and the evaluator to identify not just normal sperm but also identify sperm abnormalities consistently. Previous studies have compared various techniques for morphologic analysis of stallion sperm [[5], [6], [7], [8], [9]], using ANOVA or product-moment correlation statistical analyses. The concept of agreement, as defined by Bland & Altman method [13], warrants further consideration, because it measures the difference between techniques, and also the range of the agreement [[13], [14], [15], [16]].

This study aimed to compare the effect of 1) sperm preparation technique (EN-eosin nigrosine, Dip-Quick - DQ, PH-phase-contrast or DIC-differential interference contrast microscopy); 2) evaluator; and 3) statistical method, on the identification of sperm morphologic features in a group of stallions categorized by sperm quality (i.e., High, Moderate, and Low).