Elsevier

Theriogenology

Volume 76, Issue 4, 1 September 2011, Pages 745-750
Theriogenology

Research article
Effect of method and clinician on stallion sperm morphology evaluation

https://doi.org/10.1016/j.theriogenology.2011.04.007Get rights and content

Abstract

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.

Introduction

Sperm morphology evaluation is an essential component of semen analysis and provides the clinician invaluable information for assessing the breeding soundness of a stallion and the potential fertility of individual semen samples. Although the Society for Theriogenology (SFT) recommends the use of wet-mount semen preparations and phase-contrast or differential interference contrast microscopy for evaluation of sperm morphology in stallions [1], comparisons among different methods have apparently not been reported. Eosin/nigrosin is a stain recommended by the SFT for evaluation of bull sperm morphology [2] and is widely used, mainly because of its ease of use. In contrast, the World Health Organization (WHO) recommends the use of Papanicolaou stain for evaluation of human sperm morphology [3].

Several studies from laboratories evaluating human sperm described variations in sperm morphology results that could be attributed to technician differences [4], [5], [6], [7]. One of the main reasons for this variation was likely the lack of quality control measures, including appropriate training, continuing education, and proficiency testing. Although no similar studies have been reported, the same issues are expected to affect sperm morphology assessment in veterinary andrology laboratories.

The objectives of the present study were to determine the effect of method and clinician on stallion sperm morphology evaluation.

Section snippets

Preparation of semen samples

Sixty semen samples from 34 stallions (1 to 4 samples per stallion) were evaluated in this study. Stallions represented 12 breeds, ranged from 3 to 23 y of age, and were part of the population presented to the New Bolton Center of the University of Pennsylvania School of Veterinary Medicine for breeding soundness evaluation, fertility problems, breeding management, or semen processing for shipment or freezing. As such, this diverse population represented a wide range of fertility, from normal

Results

The proportion of sperm with “other sperm defects” (duplicate heads and tails, and teratoids) was very low (< 0.5%) and was excluded from the analysis. There were method effects (P < 0.05) on the proportions of all sperm morphology categories (Table 1). The proportion of normal sperm was greater (P < 0.05) and the proportions of acrosome, head, midpiece, and principal piece defects, and cytoplasmic droplets were less (P < 0.05) in Papanicolaou-stained samples when compared to wet-mount

Discussion

Differences in sperm morphology results among different evaluation methods can only be attributed to introduction of artifacts, poor resolution/definition of sperm structures, and unfamiliarity of the evaluator with the distinctive appearance of sperm processed by a particular method. Studies comparing methods of sperm morphology evaluation have either used only the proportion of normal sperm, which by itself is not a reliable endpoint from which to draw conclusions, or have made little effort

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