Modified diff-quick staining for canine sperm morphology

Abstract

Sperm morphology assessment requires specialized microscopes or stains. Diff-quick (DQ) is considered a universal stain that is cost-effective; however, morphological evaluation of sperm using DQ staining is poor and not encouraging. Therefore, this study investigated modifications to the DQ protocol to improve identification of morphological defects of dog sperm and compared the modified DQ techniques with eosin-nigrosin, Karras, and differential interference contrast microscopy [DIC]). One ejaculate from each of 9 dogs was used. To perform the proposed modified DQ techniques (DQ1 and DQ2), dried semen smears were fixed by immersing for 10 seconds in solution 1 of DQ and 5 minutes each in solutions II and III of the kit. After the third stain solution, slides in DQ1 were rinsed in water whereas slides in DQ2 were not rinsed but were vertically supported to facilitate stain drainage. Results suggested that the standard DQ protocol overestimated normal sperm and detached heads whereas underestimated abnormal heads and total defects compared to DIC, Karras, eosin-nigrosin, and DQ2. Acrosome abnormalities were only detectable with Karras, DIC, and DQ2. In conclusion, prolonging exposure to DQ staining solutions enhanced sensitivity in sperm morphological evaluation, and avoiding rinse as a final step in the DQ protocol improved visualization of certain acrosome defects in dog sperm. Therefore, modified DQ techniques can serve as a viable alternative for dog sperm morphology evaluation in clinical practice.