Effects of staining method and clinician experience on the evaluation of stallion sperm morphology

Evaluation of sperm morphology is part of the assessment of fertility in human and animal reproduction. Analyses can be performed using different techniques, including the use of staining methods In our prospective study, the morphology of equine sperm was evaluated using 3 staining methods Diff-Quik, eosin-nigrosin, and SpermBlue, the latter being a relatively new stain commonly used for human sperm. Our hypotheses were that (1) SpermBlue allows easier assessment of horse sperm morphology and facilitates better identification of sperm abnormalities, and (2) sperm morphology classification differs depending on the evaluator's experience. Semen was obtained from various horse breeds; 40 samples from stallions between 2 and 15 years of age were collected during the 2016 breeding season and stored in a 2% buffered formaldehyde solution until processing. For each sample, 3 semen smears were made and stained with Diff-Quik, eosin-nigrosin, and SpermBlue. All morphological parameters were then evaluated blindly using a light microscope by a novice evaluator and a more experienced evaluator. For each slide, 200 spermatozoa were examined randomly and classified according to their characteristics. For the identification of morphologically normal spermatozoa, no significant difference between evaluators was found with any of the staining methods used. In contrast, significant differences between evaluators were observed in the classification of some anomalies affecting mainly the midpiece and the tail. Poor dye fixation was observed with SpermBlue.