An In Vitro Evaluation of Biochemical Processes Involved in Lead‐Induced Changes on Ram Spermatozoa

Lead (Pb²⁺) is a toxic heavy metal which interferes with several physiological processes regulated by Ca²⁺, including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb²⁺/ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb²⁺ on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb²⁺ have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb²⁺ action. On the contrary, Pb²⁺‐incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb²⁺ + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb²⁺ in spermatozoa; thus, the enhancement of Pb²⁺ effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb²⁺ by spermatozoa. However, the increase of intracellular Pb²⁺ in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.     

Effect of Different Levels of Glycerol and Cholesterol‐Loaded Cyclodextrin on Cryosurvival of Ram Spermatozoa

This study was conducted to determine the optimum level of glycerol and cholesterol‐loaded cyclodextrin (CLC) in a Tris‐based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 10⁶ cells in Tris‐based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin–nigrosin staining) and functional membrane integrity (hypo‐osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 10⁶ sperm. Post‐thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3‐h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 10⁶ cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.     

Changes in Actin Distribution of Ram Spermatozoa under Different Experimental Conditions

The purpose of this study was to determine the presence of actin in ejaculated ram spermatozoa and the changes of localization that actin undergoes as a consequence of certain in vitro -induced physiological states. Using indirect immunofluorescence (IIF), three different patterns of staining (defined immunotypes) were established in ejaculated sperm. The three sperm immunotypes showed actin labelling in flagellum, neck and post-acrosomal area, differing on the labelling in the acrosomal region that was complete in immunotype 1, partial (frequently concentrated in the apical area, punctuate form) in immunotype 2, and totally absent in immunotype 3. The main subpopulation in ejaculate was immunotype 1 that represented 68% of total sperm, while 21% corresponded to immunotype 2 and only 10% corresponded to immunotype 3. Selection of high-quality sperm using a dextran/swim-up procedure hardly influenced the proportion of each immunotype resulting in a slight increase in type 1 sperm. Cold-shock treatment and in vitro capacitation induced a partial loss of actin labelling in the acrosomal area, whereas the ionophore-induced acrosomal exocytosis provoked a total loss of the acrosomal actin labelling, a phenomenon partially inhibited by phalloidin.     

Assessment of Fertility after Using Different Procedures to Thaw Ram Spermatozoa Frozen in Mini Straws

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In this study, fertility rates were compared after using different procedures (50°C and 70°C) to thaw ram spermatozoa frozen in mini straws. Semen from three, 1.5–2.5-year-old rams of the same breed, selected for use in an AI programme, was collected using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in 0.25-ml mini straws and frozen in a programmable freezer. Post-thaw sperm motility was assessed subjectively using a phase contrast microscope. Sperm membrane integrity was assessed with fluorescent dyes (Calcein AM/EthD-1). Statistically significant variation in the incidence of membrane integrity was found, both between rams and between freezing operations. Significant differences between the different thawing procedures used in this study were seen for membrane integrity (p < 0.01), as assessed with the fluorescent dyes (Calcein AM/EthD-1), but not for the post-thaw motility. The average fertility in this study was 39.7%, with a wide variation between freezing operations (not significant), rams (p < 0.001; 30.4, 33.3 and 64.6%) and flocks (p < 0.001, range: 14.8–61.6%). No statistically significant differences were found for the different thawing procedures, in terms of the fertility (39.0 and 40.4%, respectively) and the litter size (1.32 and 1.41, respectively). Thawing at 50°C for 9 s, instead of 70°C for 5 s, does not seem to further affect either fertility or litter size. The use of this lower temperature would facilitate the practical use of frozen–thawed ram semen under farm conditions in Sweden.     

CASA Assessment of Kinematic Parameters of Ram Spermatozoa and their Relationship to Migration Efficiency in Ruminant Cervical Mucus

Sperm motility is an indicator of male fertility because of its importance for sperm migration through the female genital tract and for gamete interaction at fertilization. This study analyses the relationship between computer assisted semen analysis (CASA) motility patterns and sperm migration of rams in ruminant cervical mucus. In experiment 1, spermatozoa extended with sperm analysis medium (SAM) and seminal plasma were compared in terms of motility. In experiment 2, 56 semen samples were collected either with artificial vagina (AV) or electroejaculator to be compared in terms of motility performance. In experiment 3, 104 ejaculates collected by AV from 26 males were analysed via the CASA system to characterize their motility patterns. In experiment 4, ejaculates from pairs of rams (20 rams in total) were simultaneously assessed for mucus migration (ovine, caprine, bovine) and motility patterns to evaluate the correlations between both parameters. Semen collected by AV and extended in SAM allows the most reliable assessment for sperm motility. Ram spermatozoa move fast and follow a linear trajectory compared with other ruminants. Continuous line velocity (VCL) and average path velocity (VAP) are the only sperm kinematic parameters that presented significant positive correlations with the ability to migrate in sheep cervical mucus (p < 0.05). Continuous line velocity, VAP, straight line velocity and linearity are highly significantly related with migration efficiency in goat cervical mucus (p < 0.01) and only lateral head displacement is negatively related to sperm migration in bovine cervical mucus (p < 0.05). These results suggest that specific kinematic parameters confer the ability of spermatozoa to colonize and migrate through epithelial mucus with different rheological properties.     

Cleaved PARP‐1, an Apoptotic Marker,can be Detected in Ram Spermatozoa

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly‐ADP‐ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly‐ADP‐ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 μm ) or betulinic acid (200 μm ). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12‐h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between‐male differences on sperm fertility.     

Evaluation of Three Different Extenders for Use in Emergency Salvaging of Epididymal Spermatozoa from a Cantabric Brown Bear

The Cantabrian brown bear (Ursus arctos) constitutes an endangered subpopulation of the European brown bear in the north of Spain. We have carried out a post‐mortem recovery of epididymal spermatozoa from a Cantabrian brown bear (7 years old, 170 kg; 30 min post‐mortem), cryopreserving those recovered from the cauda epididymis (929 × 10⁶ spermatozoa, 54% motile, 82% cytoplasmic droplets). For freezing, three extenders based on Test‐Tris‐Fructose + 4% glycerol were used: (1) 325 mOsm/kg and 10% egg yolk; (2) 430 mOsm/kg and 15% egg yolk; (3) 300 mOsm/kg, Equex‐EDTA and 20% egg yolk. After thawing, we obtained higher motility for extender 3 (31%), but extender 2 yielded the highest viability (66.9%) and mitochondrial activity (67.1%). Caffeine stimulation showed that extender two rendered the highest recovery values of post‐thawing motility with respect to the fresh sample. In conclusion, epididymal spermatozoa of brown bear can be frozen applying an extender with osmolality similar to epididymal environment.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Evaluation of Fertilizing Potential of Frozen-thawed dog Spermatozoa Diluted in ACP-106® using an In Vitro Sperm–Oocyte Interaction Assay

The aim of present study was to evaluate frozen canine semen with ACP-106 (Powder Coconut Water) using an in vitro sperm--oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106 containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106 containing 20% egg yolk and 12% glycerol. Samples were thawed at 38 degrees C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 +/- 14.8% and it was significant higher than the total motility estimated by CASA (23.0 +/- 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 +/- 3.1% and 94.3 +/- 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 +/- 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106 was efficient for maintain the in vitro fertility potential of dog spermatozoa.     

Cell Subpopulation‐related Volumetric Parameters: a Complementary Tool of the Modified Hypo‐osmotic Swelling Test on Model of Boar Spermatozoa

It is a general property of the intact animal cell to swell rapidly in response to hypo‐osmotic conditions. The modified hypo‐osmotic swelling test (HOS‐test) is an indicative test to evaluate the integrity of the plasma membrane by means of an electronic cell counter, based on the relative increase of the cell volume in response to hypo‐osmotic conditions. In this study the relationships between the osmotically induced changes of the cell volume of boar spermatozoa as determined by cell counter and the integrity of the membrane as determined by propidium iodide staining (PI) were studied. Boar sperm cell volume distributions were measured under iso‐osmotic (300 mosmolar) conditions and after a hypo‐osmotic stress (150 mosmolar). The relative volume shift of mean and modal volume were calculated as a proportion coefficient of modal and mean values of the cell volume distributions by transition from iso‐osmotic to hypo‐osmotic conditions. The volumetric parameters related to the different cell subpopulations were derived from the different peaks of cell volume distributions. PI‐staining techniques were used for comparison. The values of the volume shift and of derived percentages of the osmotically inactive cells were correlated negatively and positively, respectively (p < 0.05) with the percentage of the PI‐stained cells. This correlation indicates that a relationship exists between membrane functions of the different cell compartments (sperm head and tail) due to the circumstance that the increase of the cell volume in the HOS‐test is associated with the morphological changes in the tail and the PI‐staining is associated with the membrane integrity and permeability of the head region. The advantage of computer‐assisted volume measurement is that a large number of cells (5000–50 000 spermatozoa) can be measured and evaluated during one procedure and in a very short time. The relative volume shift is a quantitative continuous parameter characterizing the osmotic reactivity and membrane functional competence of a cell population and of subpopulations within one ejaculate. This parameter could be useful to evaluate membrane functional competence rapidly and sensitively.     

Effects of Melatonin Implants During Non‐Breeding Season on Sperm Motility and Reproductive Parameters in Rasa Aragonesa Rams

The effect of melatonin implants administered during non‐breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer‐assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona‐pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen‐thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non‐breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non‐breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.     

Comparison of Membrane‐Permeant Fluorescent Probes for Sperm Viability Assessment in the Ram

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR‐14; Hoechst‐33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR‐14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane‐affected sperm (semen treated with three cycles of freezing to ?20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma‐intact sperm determined by acridine orange and SYBR‐14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.     

Treatment of Persistent Mating‐Induced Endometritis in Mares with the Non‐Steroid Anti‐Inflammatory Drug Vedaprofen

Recently, successful treatment of mares with a history of persistent mating‐induced endometritis (PMIE) with dexamethasone has been reported. As systemic treatment of horses with glucocorticoids should be handled with caution, we tested the hypothesis that treatment with the non‐steroid anti‐inflammatory drug (NSAID) vedaprofen, an inhibitor of cyclooxygenase‐2, may have comparative, positive effects on fertility. Barren mares with a history of repeated PMIE were treated with vedaprofen (n = 8; initially 2 mg/kg bodyweight followed by 1 mg/kg orally twice daily) from 1 day before the first insemination to 1 day after ovulation or left untreated (n = 9). All mares received oxytocin (20 I.E. s.c.) thrice daily. Uterine swabs were collected for bacteriology and cytology. The day after ovulation, fluid accumulation was detected in three control mares and four treated mares (n.s.). The percentage of neutrophils in uterine cytology was significantly increased in comparison to the day before ovulation irrespective of treatment. Pregnancy was confirmed in two of nine mares in the control group and seven of eight mares in the treatment group (p < 0.05). NSAIDs may positively affect fertility in mares with a history of PMIE.     

A Systematic Review of Studies Performing the Hypo‐Osmotic Swelling Test to Evaluate the Quality of Canine Spermatozoa

The hypo‐osmotic swelling test (HOS test) is a simple and inexpensive test to evaluate the functional integrity of sperm cell membranes. According to the existing literature, its simple applicability has turned it into a valuable additional parameter to standard canine semen analysis. In the recent years, much research has been conducted in this field. The aim of this systematic review was to evaluate the quality of published literature in canine reproduction concerning the HOS test. Using two distinguished databases, 38 articles were detected and analysed subsequently according to various aspects, for example study design, population, semen sampling and implementation concerning the HOS test. Although there are numerous articles available, the diagnostic value of the HOS test remains ambiguous. Until now, neither a recognized test protocol nor reliable reference values have been defined. Most of the trials evaluated show serious methodological flaws and therefore do not permit drawing reliable conclusions. According to our results, approximately half of the studies (n = 20) included a sample size of five or less animals. None of the studies examined the inter‐ or intraobserver agreement for the HOS test. Further research is warranted including appropriate statistical methods and a sufficient number of animals to establish a standardized test protocol as well as reliable reference values. Most importantly, it is required to clarify a correlation between the HOS test and the fertilizing capacity to determine the diagnostic value of the HOS test.     

Comparison of Blood Non‐Specific Immune Parameters in Bovine Virus Diarrhoea Virus (BVDV) Persistently Infected and in Immune Heifers

Several data from different authors show that Bovine virus diarrhoea virus (BVDV) could be a key component in multiple‐etiology diseases, indeed a lower leukocytes number and their impaired functions decrease the resistance to infections. However, most of the information on the impairment of immune function during BVDV infections arise from circumstantial evidence and from experimental infection studies, and few from field data. To assess the effects of BVDV on blood cells parameters, cellular and humoral functions under field conditions, we designed a controlled study in commercial dairy herds, comparing persistent infected (PI) and healthy heifers. A total of 45 heifers were considered, the PI animals were nine, the control animals were 34, while two controls were considered as acute infected animals. The comparison of the mean values in PI calves showed a significant decrease for leukocytes and granulocytes, while platelets showed a significant increase, when compared with control animals. The total number of lymphocytes decreased not significantly in PI animals, while the proportion significantly increased. The number and proportion of monocytes was significantly reduced in PI animals, when compared with controls. The data collected on markers of cellular immunity during our study cannot be compared with the literature because there are no reference values. The presence of a persistent infection affected the cellular enzymes: NAGase, lysozyme and respiratory burst showed a large statistically significant decrease in PI animals when compared with controls. The presence of a persistent infection with BVD virus influenced blood cells number and impaired some blood cell functions. Such impairment confirms that PI animals represent a threat to the herd not only because they could spread BVDV, but also because they are more susceptible to other infectious diseases.     

Reproductive Patterns in the Non‐Breeding Season in Asinina de Miranda Jennies

This study aims to characterize the reproductive patterns in Asinina de Miranda jennies during the non‐breeding season. Reproductive activity was surveyed in 12 females, aged between 3 and 18 years old, using ultrasound and teasing with a jack. The animals were monitored from September to April, six in each consecutive year. Of these 12 females, nine showed disruption to the normal pattern of ovarian activity during the non‐breeding season. Loss of normal cyclicity included anoestrus (41.7%), silent ovulatory oestrus (25%), and persistence of corpus luteum (8.3%). Only three females maintained a regular cyclic pattern with oestrous behaviour during the non‐breeding season. Anoestrus began in early November and lasted for an average of 147 ± 28 days (113–191 days), ending near to the spring equinox. Onset of silent oestrous cycles began more erratically, between October and February. In both groups the first behavioural ovulation of the year occurred around the time of the spring equinox. Disrupted reproductive activity was preceded by a shorter oestrous cycle only in females entering anoestrus. The mean follicle size in the first ovulation of the year was larger than in the reproductive season (44.7 ± 2.45 mm vs 39.2 ± 3.60 mm) in anoestrous jennies with protracted oestrus. Though age and body condition score (BCS) were associated, changes in BCS below a threshold of four points (for anoestrus) and five points (for silent oestrus) contributed greatly to disruption of reproductive cycles. BCS in females with regular oestrous cycles during the winter season remained unchanged or exceeded five points prior to the winter solstice.     

The Accident Case—IV. The Significance and Estimation of Damage to the Central Nervous System

Abstract— The effects of mechanical injury to the brain and spinal cord are considered, special reference being given to the correlation of clinical manifestations with localization of lesions. Emphasis is placed on the importance of vascular interference in the genesis of central nervous injury.     

Short‐Term Storage and Swim‐Up Selection Do Not Affect the X/Y Ratio in Equine Spermatozoa

The standard procedure of artificial insemination with fresh equine spermatozoa involves short‐term storage (to 48 h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short‐term storage and the swim‐up procedure. We used a standard protocol, for short‐term storage (0, 24 and 48 h at 5°C) of stallion semen diluted in the commercial extender EquiPro? (Minitüb GmbH, Tiefenbach, Germany). After each set‐up storage period, the motile fraction of sperm cells was selected by the swim‐up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non‐selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1 : 1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48 h and the swim‐up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species‐related differences.