Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Manipulated sperm motility via soot nanoparticles-induced biochemical alterations in human seminal plasma

Obtaining spermatozoa with progressive motility, via postejaculatory activation with pharmacological agents such as theophylline and pentoxifylline, is crucial for the success rate of assisted reproduction in couples with severe male factor infertility. Regrettably, the possibility of premature acrosome reactions and impared oocyte function questions the practical applicability of phosphodiesterase inhibitors. The rapid development of nanotechnologies promotes the use of hydrophobic rapeseed oil soot as a non-cytotoxic biomaterial for sperm motility activation, but the scarcity of knowledge regarding the interactions of soot with components from the seminal plasma hinders the eventual commercialization of this cutting-edge approach. Aiming to eliminate this shortcoming, the current study shows for the first time how the soot nanomaterials alter the biochemistry of human seminal plasma. Upon 270 min incubation with soot nanoparticles, the activity of AST, ALT, CK, LDH and GGT enzymes in the seminal plasma of ten patients changes inversely to the registered sperm motility (i.e., lower enzyme activity, higher sperm motility and vice versa). This phenomenon is primarily related to termination of the enzymes-substrate binding or extraction of enzymes from the gametes via chemical bonding with the soot. These novel mechanisms depend on the physicochemical features of used carbon nanomaterials, revealing opportunities for predictable tuning of the sperm reproductive potential.     

Agreement between measures of total motility and membrane integrity in stallion sperm

Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and absolute difference) between TMOT and SMI was high (-0.7 and 5.6%, respectively), but decreased after 24 (10.8 and 15.1%, respectively) and 48 h (23.0 and 23.8%, respectively) of cooled storage as motility declined more rapidly than SMI. We concluded that TMOT and SMI measured separate aspects of sperm quality.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa

The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion.     

Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma

Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from “good” or “bad” freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from “good freezer” stallions (SLC-GF); iii) SLC plus pooled SP from “bad freezer” stallions (SLC-BF). After thawing, the following parameters were evaluated: chromatin integrity (DNA fragmentation index; %DFI), mitochondrial membrane potential (MMP), membrane integrity (MI), reactive oxygen species (ROS) and sperm kinematics. The %DFI was reduced (P < 0.0001) in SLC samples compared to controls. The SLC group showed a lower proportion of spermatozoa with low MMP and a higher proportion of spermatozoa with high MMP than other groups (P < 0.0001), and had lower hydrogen peroxide content than control. Sperm kinematics were not different. In conclusion, selection by SLC prior to cryopreservation improved post-thaw sperm quality; inclusion of SP from “good” and “bad” freezer stallions did not have an additional beneficial effect.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation

The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma membrane disorders (mean ± SEM 9.48 ± 0.65 and 1.62 ± 0.23% in raw and frozen-thawed sperm, respectively). However, both membrane peroxidation and protein phosphorylation were increased (P < 0.05) compared to raw semen (1.74 and 5.20-fold, respectively). There was a correlation (r = 0.73; P < 0.05) between the increase in lipid peroxidation and tyrosine phosphorylation. Seminal plasma, regardless of origin, reduced (P > 0.05) tyrosine phosphorylation present on the surface of cryopreserved sperm; however, lipid peroxidation was not significantly reduced. In conclusion, we inferred that emergence of phosphorylated proteins on the surface of cryopreserved sperm was due to increased lipid peroxidation that occurred during the freezing/thawing process; however, reduced tyrosine phosphorylation that occurred after addition of seminal plasma was triggered by other mechanisms, apparently independent from the reduction in membrane peroxidation.     

Assessment of plasma membrane and chromatin structure of sperm from transgenic and non-transgenic boars

The aim of this study was to determine the apoptotic changes and chromatin damage in non-transgenic and transgenic boars carrying the human α1,2-fucosyltransferase gene. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars both crossbreds of Polish Landrace and Large White. Two fluorescence methods were employed to measure apoptosis: an assay to assess the early changes in sperm membrane integrity using fluorophore YO-PRO-1 and an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V. The chromatin damage was assessed based on the sperm chromatin structure assay method. No significant differences in the proportion of all detected subpopulations of spermatozoa were found between TG and NTG boars. Similarly, the analysis of the chromatin structure revealed no statistical differences in the sperm chromatin damage between TG and NTG boars. In conclusion, the presence of the human α1,2-fucosyltransferase gene in the genome of TG boars did not cause any spermatogenesis process disturbances leading to the increased production of apoptotic spermatozoa. Moreover, the low level of sperm with damaged chromatin in TG boars confirms the high stability of the spermatogenesis process in the TG boars analyzed.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity

There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin integrity of SLC-selected stallion spermatozoa in 44 semen samples over time. Sperm progressive motility (P < 0.01) and the proportion with class A velocity (>50 μm/sec) were increased in samples where SP was restored, whereas the proportion with class B velocity (10 to 50 μm/sec) was decreased compared with SLC samples. However, after 24 h cold storage of treated samples, progressive motility was not different for the SP-treated groups compared with SLC, whereas chromatin damage DNA fragmentation index (%DFI) was higher. In contrast, adding SP to untreated 24 h-stored SLC samples did not affect progressive motility although it did increase the proportion of spermatozoa with class A velocity. There was individual variation between stallions whether 5% or 10% SP produced a greater increase in progressive motility. In conclusion, 5% to 10% SP can be added back to SLC-selected samples if considered necessary to optimize fertility. However, it should be added immediately before insemination rather than before storage of the sperm dose, to benefit from the transient increase in sperm progressive motility and avoid increased chromatin damage.     

Selenoprotein P in seminal fluid is a novel biomarker of sperm quality

Hepatically-derived selenoprotein P (SePP) transports selenium (Se) via blood to other tissues including the testes. Male Sepp-knockout mice are infertile. SePP-mediated Se transport to Sertoli cells is needed for supporting biosynthesis of the selenoenzyme glutathione peroxidase-4 (GPX4) in spermatozoa. GPX4 becomes a structural component of sperm midpiece during sperm maturation, and its expression correlates to semen quality. We tested whether SePP is also present in seminal plasma, potentially correlating to fertility parameters. Semen quality was assessed by sperm density, morphology and motility. SePP was measured by an immunoluminometric assay, and trace elements were determined by X-ray fluorescence spectroscopy. SePP levels were considerably lower in seminal plasma as compared to serum (0.4 ± 0.1 mg/l vs. 3.5 ± 1.0 mg/l); Se concentrations showed a similar but less pronounced difference (48.9 ± 20.7 μg/l vs. 106.7 ± 17.3 μg/l). Se and Zn correlated positively in seminal fluid but not in serum. Seminal plasma SePP concentrations were independent of serum SePP concentrations, but correlated positively to sperm density and fraction of vital sperm. SePP concentrations in seminal plasma of vasectomized men were similar to controls indicating that accessory sex glands are a testes-independent source of SePP. This notion was corroborated by histochemical analyses localizing SePP in epithelial cells of seminal vesicles. We conclude that SePP is not only involved in Se transport to testes supporting GPX4 biosynthesis but it also becomes secreted into seminal plasma, likely important to protect sperm during storage, genital tract passage and final journey.     

Comparison of sperm viability with seminal plasma metal levels

This study compares the semen levels of lead (Pb), cadmium (Cd), and aluminum (Al) in relation to live sperm in semen samples from 64 apparently healthy men. The measured levels were separated into live sperm count tertiles (<25% [18 subjects], 25–50% [26 subjects], and >50% [20 subjects]). The mean ± SD for each group was calculated, and the difference between the means of the high and low tertiles were compared by ANOVA. Significant differences were observed between the high and low live sperm groups for Pb (p < 0.01) and Al (p < 0.05), but not Cd. Spearman’s rank correlation between sperm viability and the semen plasma metal levels showed a direct relation to Mg (p < 0.05). However, there was an inverse relation to lead (p < 0.001), cadmium (p < 0.01), and aluminum (p < 0.01). There was no significant correlation between Ca and Zn. Linear regression between the live sperm counts and semen level of the three metals show that metal levels were inversely correlated with the percentage of live sperm (p < 0.001, <0.01). Apparently, the presence of these metals in the environment and in seminal plasma exerts a toxic effect on sperm.     

Colloidal centrifugation with Androcoll-E™ prolongs stallion sperm motility, viability and chromatin integrity

The objective was to investigate the changes in stallion sperm quality (sperm motility, viability, membrane integrity and chromatin integrity) occurring during cool storage, and to study the effect of sperm selection by single layer colloidal centrifugation on these parameters of sperm quality. Spermatozoa from 3 stallions (10 ejaculates, 3–4 per stallion) were selected by centrifugation through a single layer of colloid (SLC). The resulting sperm preparations and the control samples (extended but unselected semen samples) were stored at 5 °C for 48 h. Assessments of sperm quality, such as sperm motility, viability (SYBR-14/PI staining), membrane stability (Annexin-V/PI staining) and chromatin integrity, were performed on aliquots of the selected sperm preparations and unselected samples on the day of collection (3 h) and after 24 and 48 h of storage. In the SLC-selected sperm samples, sperm motility, sperm viability, proportions of spermatozoa with normal morphology and with intact chromatin were significantly better than in unselected samples (motility: 77 ± 4% vs. 64 ± 8% at 3 h; P < 0.001; viability: 79.5 ± 9% vs. 64.7 ± 9%, P < 0.001; normal morphology 89 ± 6% vs. 69 ± 9%; chromatin integrity DFI 11.3 ± 5% vs. 22.1 ± 10%). Membrane stability, however, was not different in the SLC-selected and unselected samples (74.6 ± 8% vs. 69.3 ± 8%). The deterioration seen in sperm quality in the unselected samples was prevented by SLC, so that sperm viability, membrane stability and chromatin integrity were unchanged in the selected samples by 48 h compared to 3 h (P < 0.001), whereas the unselected samples were significantly worse by 48 h (P < 0.001). Furthermore, it should be possible to send an aliquot of a normal insemination dose (i.e. unselected spermatozoa) overnight to a reference laboratory for analysis of both plasma membrane and chromatin integrity. In conclusion, centrifugation of stallion spermatozoa through a single layer of colloid is a useful technique for selecting the best spermatozoa from an ejaculate and, moreover, sperm quality is maintained during storage.     

Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma.

A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCl containing 0.1 mM DTT and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration-dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.     

Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.     

Effect of method and clinician on stallion sperm morphology evaluation

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.