Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

Effect of seminal plasma concentration and various extenders on postthaw motility and glass wool-Sephadex filtration of cryopreserved stallion semen

OBJECTIVE: To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. SAMPLE POPULATION: 7 stallions from which we collected > or = 3 ejaculates/stallion. PROCEDURES: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or dilution rate could improve ability of such semen to survive freeze-thaw procedures. RESULTS: Use of Kenney extender as the centrifugation extender significantly improved postthaw motility and GWS filtration, compared with glucose-EDTA. Extending semen at a dilution of 1:3 was significantly better than 1:1 for both motility and GWS filtration. In addition, including seminal plasma at a concentration of 5% in the cryopreserved semen resulted in significantly higher yield of spermatozoa after GWS filtration, compared with complete removal of SP or use of seminal plasma at 25%. Lastly, semen with poor postthaw quality had significantly improved postthaw quality in regard to motility and GWS filtration when semen was frozen with seminal plasma at a concentration of 5%, compared with semen frozen with seminal plasma at a concentration of 25%. CONCLUSIONS AND CLINICAL RELEVANCE: Use of Kenney extender at a high dilution (> or = 1:3) immediately after collection of semen can improve postthaw quality of frozen stallion semen.     

Upregulation of CRISP-3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage

For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expression in SP with either positive or negative effects on sperm survival during storage was studied using two-dimensional differential gel electrophoresis and liquid chromatography–mass spectrometry. Lower sperm chromatin integrity was associated with upregulation of the proteins kallikrein, CRISP-3 and HSP-1, while higher chromatin integrity was associated with upregulation of TIMP-2. In the sperm-rich fractions, kallikrein and CRISP-3 differed significantly between SP samples with differing effects on sperm chromatin integrity. In the sperm-poor fractions, TIMP-2 and HSP-1 differed significantly between the two SP groups. Differences in the seminal plasma proteome are associated with sperm longevity during cooled storage.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Epigallocatechin‐3‐gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin‐3‐gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.     

Alkaline and Acid Phosphatase, β‐Glucuronidase and Electrolyte Levels in Fractionated Stallion Ejaculates

Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β‐glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections were done using a computer‐controlled phantom that collects the ejaculatory jets separately in five cups. The cups with the highest (HIGH) and the lowest (LOW) sperm concentration were analysed. In Trial I, semen was collected from three reproductively normal stallions. In Trial II, ejaculates of two reproductively normal stallions were compared to those of two subfertile stallions. In Trial III, semen was collected from seven stallions with varying reproductive history. The sperm‐rich fractions contained the highest levels of AP, ACP, BG and inorganic phosphate, and the values were positively correlated to the sperm concentration. Significant differences between the subfertile and the fertile stallions pairs in HIGH : LOW ratios were found in Pi and Cl concentrations. The highest concentrations of Ca and Mg were found in the last fractions with low sperm concentrations, with no significant differences between the fertile and the subfertile stallion pairs. The concentrations of K, Na and Cl were similar in HIGH and LOW fractions and in whole ejaculate samples. Pre‐sperm fluid contained the highest concentrations of Na and Cl. Some of the possible variation in storage tolerance between ejaculates and ejaculatory fractions could perhaps be explained by differences in the composition of SP.     

Effect of seminal plasma fractions on stallion sperm survival after cooled storage

This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm-rich fractions (SRF) or sperm-poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 x 10(6) sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions' second (SP-SRF) or last cup (SP-SPF). Skim milk extender (K) and skim milk extender supplemented with modified Tyrode's medium (KMT) were used as control treatments. After a 24-h storage period in a transport container, spermatozoa were evaluated for motion characteristics and plasma membrane integrity by calcein acetoxymethyl (AM)/propidium iodide staining. The percentage of spermatozoa with intact plasma membranes after storage was lower in SP-SRF than in SP-SPF, and the highest in K (P < 0.05). Progressive motility (PMOT) was lower for sperm stored in SP-SRF than for sperm stored in SP-SPF (P < 0.05), but there was no significant difference in total motility (TMOT). Sperm stored in KMT (P < 0.05) registered the highest TMOT and PMOT percentages. Osmolarity was significantly higher and pH lower in K than in KMT or SP. Treatment with SP-SPF from three stallions benefited the PMOT of sperm from one stallion. These preliminary findings suggest that SP from SRFs may be more harmful during storage than SP from SPFs. Removal of SP improves sperm survival in KMT extender, and exchanging SP between stallions seems to influence sperm survival.     

Supplemented stallion seminal plasma can improve impaired motility due to the dilution effect in chilled Asian elephant sperm

The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris‐glucose‐egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm.     

Nickel seminal concentrations in various animals and correlation to spermatozoa quality

In this study, the concentration of nickel in stallion, bull, ram, boar and fox semen, and its relation with spermatozoa quality was analyzed. The concentration of nickel in semen was 0.20 mg kg(-1) in stallion, 0.12 mg kg(-1) in bull, 0.31 mg kg(-1) in ram, 0.06 mg kg(-1) in boar and 0.36 mg kg(-1) in fox. Seminal nickel concentration was significantly higher (P < 0.05) in foxes than that in bulls and significantly higher (P < 0.01) in rams and foxes in comparison with boars. Evaluation of total pathological spermatozoa revealed the highest number in stallions followed by rams, bulls, boars and foxes. In bull, ram and boar semen, separated flagellum, flagellum torso and knob-twisted flagellum were predominant. Knob-twisted flagellum, separated flagellum and flagellum torso were found in increased number in stallion semen and broken flagellum in fox semen. Correlation analysis in bulls indicated a high positive correlation between seminal nickel and separated flagellum (r = 0.76) and medium positive correlation between nickel and flagellum torso (r = 0.62), and in rams a high positive correlation between nickel and separated flagellum (r = 0.77). Medium positive correlation was found between nickel and separated flagellum (r = 0.43) and between nickel and other pathological spermatozoa (r = 0.45) in boars.     

Applicability of a New Cell Culture Device for Cooled‐Storage of Stallion Semen

A new device for storage and shipping of cell cultures – the Petaka G3 cell management device – was tested for its applicability for cooled‐storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg / l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15°C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer‐assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15°C than at 5°C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled‐storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.     

Characterization of semen collected by pharmacologically induced ejaculation from a stallion with seminal vesiculitis

The present study compared the quality of sperm collected by artificial vagina or pharmacologically induced ejaculation from a 10-year-old thoroughbred stallion with seminal vesiculitis. The pharmacological protocol involved intravenous administration of detomidine (0.01 mg/kg) and oxytocin (20 IU) and successfully induced ejaculation in all attempts of semen collection. Sperm motility, plasma membrane and acrosome integrity (PMAI), reactive oxygen species (ROS) levels, polymorphonuclear neutrophil (PMN) percentage, and bacterial profiles of fresh and cooled semen (5°C for 24 hr) were evaluated. Semen obtained by the pharmacological method presented reduced seminal volume, decreased PMN percentage and superior sperm motility in cooled samples. Moreover, higher PMAI and lower ROS levels were observed in semen collected by the pharmacological method. Therefore, pharmacologically induced ejaculation is an alternative to obtain semen with minimal contamination and with sperm of superior quality and longevity from stallions with seminal vesiculitis.     

Removal of Seminal Plasma Enhances Membrane Stability on Fresh and Cooled Stallion Spermatozoa

Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some ‘poor cooler’ stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4°C: motility, plasma membrane integrity as evaluated by hypo‐osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty‐six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.     

Variation among stallions in sperm quality after single layer centrifugation

Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48 hr using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all timepoints, especially chromatin integrity and MI (p < .0001 for both), and some categories of ROS production (e.g. proportion of live hydrogen peroxide negative spermatozoa, p < .0001), but the degree of improvement varied among stallions and type of ROS (p < .05–p < .0001). Total and progressive motility were also better in SLC samples than in CON at 24 and 48 hr (p < .0001), although the effect on sperm kinematics varied. The interaction of treatment, time and stallion was not significant. In conclusion, sperm quality was better in SLC samples than in CON, although there was considerable individual variation among stallions. The improvement in sperm quality, particularly in chromatin integrity, was clearly beneficial, and therefore the use of this technique would be warranted for all stallion semen samples.     

Implication of FKBP6 for Male Fertility in Horses

In stallions, impaired acrosome reaction (IAR) may often cause subfertility. Single nucleotide polymorphisms (SNPs) within FK506‐binding protein (FKBP6) seem to be associated with IAR in stallions. However, their effect on stallion fertility has not yet been quantified. Using whole‐genome sequence data of seven stallions, we searched FKBP6 for mutations to perform an association study in Hanoverian stallions with estimated breeding values for the paternal component of the pregnancy rate per oestrus cycle (EBV‐PAT) as target trait. Genotyping five exonic mutations within FKBP6 revealed a significant association of the SNP g.11040379C>A (p.167H>N) with EBV‐PAT in 216 Hanoverian stallions. The difference among the two homozygous genotypes was 7.62% in EBV‐PAT, corresponding to one standard deviation of EBV‐PAT. In conclusion, in Hanoverian stallions, the FKBP6‐associated SNP g.11040379C>A confers higher conception rates in A/A homozygous and lower conception rates in C/C homozygous Hanoverian stallions. Thus, an FKBP6‐associated missense mutation is significantly associated with stallion fertility.     

Effect of Post‐Thaw Addition of Seminal Plasma on Motility,Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.     

Seminal Characteristics in Spanish Purebred Stallions: A Retrospective Study

In this work, we studied retrospectively the influence of several factors on semen quality of the Spanish Purebred (SPB) stallion. Among these factors, we considered stallion age, season, and interval between two consecutive collections. The semen was collected from 11 SPB stallions (634 ejaculates). Semen quality was evaluated based on volume, concentration, and motility of the ejaculates. Results showed that the semen collected in autumn and winter was more concentrated in comparison with that collected in spring and summer, when the volume of ejaculate was the highest. Those ejaculates collected from younger stallions were characterized by having the smallest volume and the highest sperm concentration. When incorporating an SPB stallion into a program of artificial insemination, it is important to take into account all of these factors to achieve the maximum reproductive potential of the animal.     

Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Retrograde ejaculation in a stallion

Retrograde ejaculation was diagnosed in a 10-year-old Arabian stallion. Despite behavioral signs consistent with ejaculation, the collection receptacle of an artificial vagina remained devoid of semen on numerous occasions. Catheterization of the urinary bladder yielded large numbers of spermatozoa, even when an ejaculate was obtained, whereas low numbers (< 1 X 10(6)/ml) of spermatozoa are found in the bladder of clinically normal stallions after ejaculation. Endoscopic examination of the urethra, seminal colliculus, and bladder failed to reveal abnormalities. Medical treatment with imipramine hydrochloride apparently resulted in improvement initially, but was not curative. Further diagnostic and treatment measures were declined and the stallion was castrated. For stallions that seemingly fail to ejaculate or for ejaculates that contain lower seminal volumes or numbers of spermatozoa than expected, obtaining a urine sample after ejaculation via bladder catheterization is a simple diagnostic procedure that may be used to investigate the possibility of retrograde ejaculation.