Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Novel characterization of the HSPA2-stabilizing protein BAG6 in human spermatozoa.

While a large cohort of sperm surface receptors underpin sperm-oocyte adhesion processes, our recent work has revealed that the molecular chaperone Heat Shock Protein A2 (HSPA2) is a key regulator of zona pellucida-receptor complex assembly in our own species. Indeed, in the infertile population, spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. While the mechanisms behind this protein deficiency are under consideration, BCL2-associated athanogene 6 (BAG6) has been identified as a key regulator of HSPA2 stability in mouse germ cells. However, in the human, the presence of BAG family proteins remains completely uncharacterized. Consequently, this study aimed to determine the presence of BAG6 in human sperm cells and to characterize its putative interaction with HSPA2 throughout sperm cell development. BAG6 was shown to co-localize with HSPA2 in human testicular germ cells and epididymal spermatozoa. Similarly, BAG6 was identified in the equatorial region of non-capacitated spermatozoa but underwent a marked relocation to the anterior region of the head upon the induction of capacitation in these cells. Protein-protein interaction assays revealed the stable interaction of BAG6 and HSPA2 proteins in mature spermatozoa. Furthermore, examination of the spermatozoa of infertile men with zona pellucida binding defects, related to a lack of HSPA2, revealed a concomitant deficiency in BAG6 protein expression. In view of the findings described in this study, we propose that BAG6 is likely a key regulator of HSPA2 stability/function in human germ cells. Moreover, its under-representation in spermatozoa with zona pellucida binding deficiency suggests that BAG6 may be an important candidate to study for a further understanding of male idiopathic infertility.

In situ viability detection assays induce heat-shock protein 70 expression in spermatozoa without affecting the chromatin integrity.

To differentiate dead spermatozoa from viable but immotile spermatozoa, several techniques are being used during ICSI. As processed spermatozoa from poor-quality ejaculate are confronted with a higher risk of experiencing stress on exposure to altered osmotic conditions or chemicals, this study was undertaken to determine the expression of stress response gene Hsp70 and chromatin integrity in spermatozoa subjected to in situ viability assays such as hypo-osmotic swelling (HOS) test, modified hypo-osmotic swelling (M-HOS) test and pentoxifylline in 25 fresh and frozen-thawed asthenozoospermic ejaculates. RT-PCR and immunofluorescence detection of Hsp70 were performed to elucidate the expression and localisation of Hsp70 in spermatozoa, whereas DNA fragmentation analysis was performed by sperm chromatin dispersion assay. Exposure of fresh and frozen-thawed asthenozoospermic spermatozoa to M-HOS and pentoxifylline significantly increased Hsp70 expression as evidenced by increased RNA expression and immunolocalisation of Hsp70 protein in sperm head (P < 0.05-0.001). However, chromatin integrity was not significantly affected in any groups until 6 h of post-exposure time period. Our results suggest that conventional HOS may be preferred for the in situ detection of the viability as there was no immediate stress response and chromatin instability in the exposed spermatozoa.

Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation.

The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.

SEPT12-microtubule complexes are required for sperm head and tail formation.

The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/- chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and ß-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and ß-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and ß-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and ß-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and ß-tubulins, in SEPTIN12+/+/+/- chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.

Analysis of the sperm head protein profiles in fertile men: consistency across time in the levels of expression of heat shock proteins and peroxiredoxins.

We investigated the identity and quantitative variations of proteins extracted from human sperm heads using a label-free Gel-MS approach. Sperm samples were obtained from three men with high sperm counts at three different time points. This design allowed us to analyse intra-individual and inter-individual variations of the human sperm head proteome. Each time point was analyzed in triplicate to minimize any background artifactual effects of the methodology on the variation analyses. Intra-individual analysis using the spectral counting method revealed that the expression levels of 90% of the common proteins identified in three samples collected at various time-points, separated by several months, had a coefficient of variation of less than 0.5 for each man. Across individuals, the expression level of more than 80% of the proteins had a CV under 0.7. Interestingly, 83 common proteins were found within the core proteome as defined by the intra- and inter-variation analyses set criteria (CV<0.7). Some of these uniformly expressed proteins were chaperones, peroxiredoxins, isomerases, and cytoskeletal proteins. Although there is a significant level of inter-individual variation in the protein profiles of human sperm heads even in a well-defined group of men with high sperm counts, the consistent expression levels of a wide range of proteins points to their essential role during spermatogenesis.

Nanoscale differences in the shape and size of X and Y chromosome-bearing bovine sperm heads assessed by atomic force microscopy.

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing.

Measuring the regulation of dynein activity during flagellar motility.

Flagellar and ciliary motility are driven by the activity of dynein, which produces microtubule sliding within the axonemes. Our goal is to understand how dynein motile activity is regulated to produce the characteristic oscillatory movement of flagella. Analysis of various parameters, such as frequency and shear angle in beating flagella, is important for understanding the time-dependent changes of microtubule sliding amounts along the flagellum. Demembranated flagella can be reactivated in a wide range of ATP concentrations (from 2 µM to several mM) and the beat frequency increases with an increase in ATP. By imposed vibration of a micropipette that caught a sperm head by suction, however, the oscillatory motion can be modulated so as to synchronize to the vibration frequency over a range of 20-70Hz at 2mM ATP. The time-averaged sliding velocity calculated as a product of shear angle and vibration frequency decreases when the imposed frequency is below the undriven flagellar beat frequency, but at higher imposed frequencies, it remains constant. In addition to the role of ATP, the mechanical force of bending is involved in the activation of dynein. In elastase-treated axonemes, bending-dependent regulation of microtubule sliding is achieved. This chapter provides an overview of several approaches, using sea urchin sperm flagella, to studying the measurements in the regulation of dynein activity with or without mechanical force.

A novel in vitro sperm head decondensation protocol for rapid flow cytometric measurement of deoxyribonucleic acid content.

OBJECTIVE: To set up a novel protocol of sperm head in vitro decondensation that obviates the problematic effect of the variable degree of sperm chromatin packaging on DNA staining needed for flow cytometric analysis. DESIGN: Development of a new cytofluorimetric assay. SETTING: University laboratory. PATIENT(S): Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences, Università Politecnica delle Marche. INTERVENTION(S): Setup of the novel in vitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry to measure DNA content. MAIN OUTCOME MEASURE(S): Mean fluorescent channel, DNA content, percentage diploid sperm. RESULT(S): Native nondecondensed fluorochrome-labeled sperm show significant under-staining, resulting in an underestimated C-value (approximately 1.4 pg). This protocol ensures stoichiometric staining of sperm DNA, which becomes fully reachable by fluorescent probes and makes the diploid (7.12 pg) over haploid (3.56 pg) sperm frequency quantification easier. CONCLUSION(S): This study establishes a simple method for in vitro sperm head decondensation, which allows accurate detection of the real sperm DNA content.

A caged progesterone analog alters intracellular Ca2+ and flagellar bending in human sperm.

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.

Uneven distribution of ceramides, sphingomyelins and glycerophospholipids between heads and tails of rat spermatozoa.

Previous work showed that rat germ cells and spermatozoa contain ceramides and sphingomyelins with high proportions of nonhydroxy and 2-hydroxy (2-OH) polyunsaturated fatty acids (PUFA) with very long chains (VLCPUFA). The aim of this study was to assess how these lipids are distributed between the heads and tails of mature spermatozoa in comparison with other membrane lipid classes. In addition to quantitative differences due to the fact that these gametes have a long, voluminous tail and a minute head, several compositional dissimilarities emerged between these two regions. The total cholesterol/total phospholipid ratio, the choline/ethanolamine glycerophospholipid (ChoGpl/EtnGpl) ratio, and the proportion of plasmalogens within these two classes, were much larger in the head than in the tail. Whereas EtnGpl was rich in 22:5n-6 in both regions, ChoGpl had plenty of 22:4n-9, especially in the heads. An important proportion of the head EtnGpl- 22:5n-6 and ChoGpl 22:4n-9 was in plasmenyl- (rather than in phosphatidyl-) subclasses. The heads concentrated all of the sphingomyelin species with nonhydroxy- and 2-OH VLCPUFA, and the tails most of the saturated fatty acids that are present in total sperm sphingomyelin. Unexpectedly, virtually all of the abundant spermatozoal ceramides, predominantly made up by species with 2-OH VLCPUFA, was located in the tail. The fact that intact rat spermatozoa constitutively have much more VLCPUFA-containing ceramide than sphingomyelin is explained by the present findings, since the former are mostly lipids of the large tail while the latter mostly collect in the small head.

Characterization and subcellular localization of Tektin 3 in rat spermatozoa.

Mammalian sperm flagella have filament-forming Tektin proteins (Tektin 1-5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S-EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the peri-axonemal component and not directly associated with axonemal tubulins. Resistance to S-EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre-embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome-related events, such as the acrosome reaction or sperm-egg fusion.

Proteomic and functional analysis of human sperm detergent resistant membranes.

Mammalian spermatozoa attain the ability to fertilize an oocyte as they negotiate the female reproductive tract. This acquisition of functional competence is preceded by an intricate cascade of biochemical and functional changes collectively known as "capacitation." Among the universal correlates of the capacitation process is a remarkable remodeling of the lipid and protein architecture of the sperm plasma membrane. While the mechanisms that underpin this dynamic reorganization remain enigmatic, emerging evidence has raised the prospect that it may be coordinated, in part, by specialized membrane microdomains, or rafts. In the present study we have demonstrated that human spermatozoa express recognized markers of membrane rafts. Further, upon depletion of membrane cholesterol through either physiological (capacitation) or pharmacological (methyl-ß-cyclodextrin) intervention, these membrane rafts appear to undergo a polarized redistribution to the peri-acrosomal region of the sperm head. This finding encourages speculation that membrane rafts represent platforms for the organization of proteins involved in sperm-oocyte interactions. Support for this notion rests with the demonstration that membrane rafts isolated on the basis of their biochemical composition in the form of detergent resistant membranes (DRMs), possess the ability to adhere to homologous zona pellucidae. Furthermore a comprehensive proteomic analysis of the DRMs identified a number of proteins known for their affinity for the zona pellucida in addition to other candidates putatively involved in the mediation of downstream binding and/or fusion with the oolemma. Collectively these data afford novel insights into the subcellular localization and potential functions of membrane rafts in human spermatozoa.

Proteomic profiling reveals compartment-specific, novel functions of ascidian sperm proteins.

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X-100-soluble and -insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS-PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non-redundant proteins were identified by mass spectrometry (MALDI-TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm-specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca(2+)-binding proteins in axonemes, localization of testis-specific serine/threonine kinase, and the presence of G-protein coupled signaling and ubiquitin pathway in sperm flagella.

Hydrophobic silicone elastomer chamber for recording trajectories of motile porcine sperms without adsorption.

Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P<0.05) and ALHD (P<0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation; instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels.

X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis.

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular, and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and colocalized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.2 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis.

Temporal deposition and spatial distribution of cytoskeletal proteins in the sperm head of an Australian rodent.

The Australian murine rodent, the plains mouse (Pseudomys australis), possesses a highly complex sperm head, in which there are, in addition to an apical hook, two ventral processes that extend from its upper concave surface. The present study set out to determine the temporal deposition and distribution of the proteins within these structures during late spermiogenesis by light and electron microscopy using various antibodies to bull and laboratory rat sperm-head cytoskeletal proteins. The findings show that there are two phases of protein deposition. In the first phase, perinuclear theca proteins are deposited at the base of the ventral processes around the acrosomal extensions of the developing spermatids. In the second phase, as the ventral processes expand, actin and then perforatorial proteins are laid down during which time the processes become progressively more bilaterally flattened. These various proteins are moulded together to give rise to the two very large cytoskeletal structures that extend from the upper concave surface of the sperm head. They may be involved in binding the spermatozoon to the outer surface of the zona pellucida and/or in aiding the spermatozoon in zona penetration at the time of fertilisation.

Sperm competition: linking form to function.

BACKGROUND: Using information from physics, biomechanics and evolutionary biology, we explore the implications of physical constraints on sperm performance, and review empirical evidence for links between sperm length and sperm competition (where two or more males compete to fertilize a female's eggs). A common theme in the literature on sperm competition is that selection for increased sperm performance in polyandrous species will favour the evolution of longer, and therefore faster swimming, sperm. This argument is based on the common assumption that sperm swimming velocity is directly related to sperm length, due to the increased thrust produced by longer flagella. RESULTS: We critically evaluate the evidence for links between sperm morphology and swimming speed, and draw on cross-disciplinary studies to show that the assumption that velocity is directly related to sperm length will rarely be satisfied in the microscopic world in which sperm operate. CONCLUSION: We show that increased sperm length is unlikely to be driven by selection for increased swimming speed, and that the relative lengths of a sperm's constituent parts, rather than their absolute lengths, are likely to be the target of selection. All else being equal, we suggest that a simple measure of the ratio of head to tail length should be used to assess the possible link between morphology and speed. However, this is most likely to be the case for external fertilizers in which females have relatively limited opportunity to influence a sperm's motility.

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility.

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa.

This study was designed to compare the performance of the kits Diff-Quick, Hemacolor and Spermac for staining the spermatozoa of rainbow trout. Automated sperm morphology analysis (ASMA) was performed using two image analysis programs to determine the sperm measurements: head size (length, width, area and perimeter), shape (ellipticity, rugosity, elongation and regularity) and tail length. Diff-Quick was found to be the best procedure for staining the trout spermatozoa. The use of this method rendered the highest number of cells correctly analyzed, and provided good colour intensity and contrast of the sperm head. No differences among the methods were detected in terms of tail length measurements. Mean values established using Diff-Quick for the main morphometric variables were: head length 2.93+/-0.13 microm; head width 2.33+/-0.15 microm and tail length 34.16+/-1.66 microm. Based on these findings, we recommend the Diff-Quick staining kit for its accurate and reproducible morphometric results. Notwithstanding, when analyzing the sperm tail of the rainbow trout, the Spermac method offers improved contrast.