SpermBlue † : A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis G van der Horst, L Maree Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa Accepted March 30, 2009 Abstract Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue † fixative and SpermBlue † stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue † fixative or by adding 1 ml SpermBlue † fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue † stain or adding four drops of SpermBlue † stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX † or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue † staining process. SpermBlue † stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer † ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600  or 1000  magnification for most of the species studied. Key words: acrosome, ASMA, head, human, invertebrates, mammals, midpiece, morphology, primates, sperm, staining, tail, vertebrates Sperm morphology assessment is one of the most important criteria for determining the quality of a semen sample for humans and animals (Gra vance et al. 1998, WHO 1999, Henkel et al. 2007, Van der Horst et al. 2009). Moreover, many claims ha ve been made about the relation between sperm morphology assessment and fertility (Enginsu et al. 1991, Menkveld et al. 2003). Accordingly, many stains and staining combinations ha ve been employed to determine the percentage of sperm with normal morphology. The accuracy of sperm morphology assessment depends on careful preparation, fixation, and staining of sperm (Garcı ´a-Herreros et al. 2006), because these procedures can affect sperm dimensions significantly (Meschede et al. 1993, Gago et al. 1998, Hidalgo et al. 2006, Lukaszewicz et al. 2008). Thus, one of the criteria for a staining method should be that the processes involved cause as little change to sperm morphology as possible. Address for correspondence: Gerhard van der Horst, Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa. E-mail: gvdhorst@ uwc.ac.za – Biological Stain Commission Biotechnic & Histochemistry 2009, 110, iFirst article. DOI:10.1080/10520290902984274 1