Usefulness of Dip Quick Stain in Evaluating Sperm Morphology in Stallions

Dip Quick is an easy and practical method of staining equine spermatozoa for morphology evaluation. Prolonged time of staining (30 minutes in each solution) is advisable for best results.

1. Introduction

A variety of simple stains are used in veterinary practice to evaluate cytological samples. Commonly, Romanowsky-type, polychromic stains are used to prepare cytology slides. These stains use 3 colors (red, blue, purple) to produce variations in cell architecture that are easily detected using brightfield microscopy.¹  In veterinary practice, DiffQuick or Dip Quick stains are used because they are readily available, simple, and inexpensive. They are used for staining blood and bone marrow smears, vaginal cells, and spermatozoa in dogs.^2,3  The goal of this study was to investigate a usefulness of Dip Quick stain for evaluating sperm morphology in stallions when compared with more traditional, phase-contrast and eosin-nigrosin (E-N) methods.

2. Materials and Methods

Six mature, Miniature horse stallions (7 to 12 years old) were used in this study. Stallions were kept in individual pens made of galvanized panels (48 x 36 feet) placed in a large pasture located on the premises of the College of Veterinary Medicine, University of Florida. The horses had ad libitum access to water and were supplemented with hay and grain. This project was approved by the Institutional Animal Care and Use Committee of the University of Florida. 

Four ejaculates were obtained from each stallion on 4 consecutive days (24 ejaculates total). Semen was collected using a dummy mount and an artificial vagina fitted with a disposable liner.^a The gel fraction was separated from semen, using a nylon mesh filter.^b A small aliquot of semen from each ejaculate was fixed in buffered 10% formalin solution^c for wet-mount preparations to be evaluated with phasecontrast microscopy (Phase). Raw semen was used for stained slides to be evaluated with bright-field microscopy. Four slides were prepared from each ejaculate. For E-N staining, a droplet of raw semen was mixed with a droplet of stain^d with a wooden applicator. Using a second clean slide, a thin smear was made of the mixed solutions. To prepare Dip Quick slides (n = 3), a droplet of raw semen was smeared on a clean slide, using a second clean slide. The preparation was allowed to dry fully before staining. Dried slides were stained using 3 staining times: exposure of a slide to each stain solution^e for 5 minutes (DQ-5); exposure of a slide to each stain solution for 15 minutes (DQ-15); and exposure of a slide to each stain solution for 30 minutes (DQ-30). The slides were rinsed in distilled water and air dried again. Dip Quick stained slides were coded (A, B, C), to blind evaluators to preparation method. Morphologic evaluation of all slides was conducted by the same investigator using x 1000 magnification and phase-contrast microscopy for wet-mounts. Bright-field microscopy was used for stained slides.^f A total of 100 individual spermatozoa was counted in each slide to determine the percentages of normal spermatozoa and percentages of all identifiable morphological abnormalities. Dip Quick–stained slides were evaluated by a second investigator for subjective assessment of staining quality. Slides from each ejaculate were ranked, using a rank score of 1 to 3, with 1 being the highest score and 3 being the lowest score.

Statistical analysis was performed using the analytical software package.^g Sperm morphology results, obtained using 5 different methods (Phase, E-N, DQ-5, DQ-15, DQ-30), were compared using 1-way ANOVA. The degrees of linear correlations between all methods were tested using a Pearson product-correlation procedure. Ranks were compared between different Dip Quick staining times using Kruskal-Wallis 1-way nonparametric ANOVA, followed by an all-pairwise comparisons test. Significance was set at P < 0.05.

3. Results

Differences were not detected (P > 0.05) for percentages of normal spermatozoa or spermatozoa with various morphological defects between evaluation methods, including different staining times (Table 1). Furthermore, percentages of normal spermatozoa and spermatozoa with specific morphological defects were significantly correlated (P < 0.05) between different evaluation methods (Table 2).

Slides stained with DQ-30 were ranked first, whereas slides stained with DQ-15 were ranked second in the majority of samples (20/24); slides stained with DQ-5 were always ranked last. Differences in ranks were significant (P < 0.05), with DQ-30 being ranked highest and DQ-5 being ranked lowest. Furthermore, a second investigator observed that only slides stained with DQ-30 had spermatozoa with a well-stained acrosomal region (Figs. 1 and 2).
 

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