Measurement of Concentration and Viability in Stallion Sperm

Sperm concentration and sperm membrane intactness (SMI) or viability are two measures of sperm quality that provide important but different information about a stallion's reproductive capability. Sperm concentration is a measure that, by itself, informs little about the reproductive status of either the stallion or the ejaculate. Nevertheless, it is part of the product, along with semen volume, that determines total sperm number. The correct calculation of total sperm number directly affects the number of mares a stallion can breed and therefore, fertility. If either sperm concentration or semen volume is incorrectly measured, both the number of mares that a stallion can breed and the fertility of those breedings are affected. Although considerable between-stallion variation exists, sperm concentration, semen volume and total sperm number tend to be seasonal and vary with ejaculation frequency.     

Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality

This study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8°C), the other in a styrofoam box with ice (+3°C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4°C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 ± 0.6°C and 8.6 ± 2.5°C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3°C) or higher (+8°C) temperature than +5°C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions.     

干旱胁迫对委陵菜膜脂过氧化作用及保护酶活性的影响

用聚乙二醇模拟干旱胁迫,研究了干旱胁迫对5种委陵菜属植物膜脂过氧化作用及保护酶活性的影响。结果表明:随着干旱胁迫程度的加重5种委陵菜的MDA含量呈递增趋势,其中伏委陵菜与三叶委陵菜的增幅较大,表明其膜脂抗过氧化能力最弱,细胞被破坏的程度最大,抗旱能力最弱。SOD、POD、CAT活性随着干旱胁迫程度的增加呈现先上升后下降的变化趋势,表明在轻度干旱胁迫下5种委陵菜均能启动保护酶系统提高抗性,但随着胁迫程度增加,其保护酶系统平衡遭到破坏,加剧膜脂过氧化而造成膜损伤。     

Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Effect of Caffeine and Pentoxifylline Added Before or After Cooling on Sperm Characteristics of Stallion Sperm

Different additives have been tested in cooled stallion sperm, in order to maintain sperm quality and to ameliorate the decrease in sperm fertility potential. In several species, caffeine and pentoxifylline promote sperm motility by increasing energy production. We evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or after cooling. Three ejaculates from five stallions each were processed and resuspended in skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM), or both additives combined were included to sperm before or after cooling (4°C for 24 hours). Cooled sperm were incubated at 37°C and evaluated at 0, 30, 60, and 120 minutes for motility, morphology, viability (flow cytometry), and membrane functionality (hypo-osmotic swelling test). Results were analyzed by two-factor mixed model for repeated measures and Tukey comparisons. As main effects, the caffeine and pentoxifylline affected significantly motility and kinematic parameters, without interaction between treatment and incubation after cooling. No differences were observed whether the additives were added prior or after cooling. Pentoxifylline added after cooling reduced significantly motility during incubation, but with higher values at 30 minutes. We detected a decrease in morphologically normal sperm (P < .0001), caused by an increase of tail defects (P < .003) in the presence of both additives. Viability and membrane functionality were also significantly impaired by additives. Pentoxifylline when added after cooling improved sperm motility and kinematic parameters for a short period of time. However, sperm characteristic related to fertility potential was compromised after a prolonged exposure to caffeine or pentoxifylline.     

Sperm Susceptibility to Oxidative Stress in the Retuertas Endangered Horse

In order to determine whether the high level of inbreeding of the Retuertas endangered Horses may increase the vulnerability to oxidative stress of the sperm, the susceptibility to lipid peroxidation (LPO) of sperm membranes was evaluated in cooled extended ejaculates. Extended ejaculates (INRA 96) from four Retuertas horses and nine ejaculates from pure Spanish horses were received within 24 hours of collection at our laboratory. An aliquot was analyzed and served as time 0 control. The rest of the ejaculates were split into two additional aliquots: the first aliquot served as an incubation control and the second aliquot received an oxidative insult (Fe₂SO₄). Membrane LPO, motility parameters, membrane integrity, and mitochondrial membrane potential were evaluated. The oxidative insult induced a significant increase in the percentage of sperm with LPO in the Retuertas horses (P < .05). Total and progressive motility were not affected by the oxidative insult in both groups. However, curvilinear velocity (VCL), average velocity (VAP), straightness index (STR), and amplitude of lateral head displacement (ALH) were significantly affected by the treatment (P < .05) in the Retuertas horse population. The membrane integrity of sperm in this group was also affected: the percentage of sperm with intact membranes decreased (57.31% ± 7.76% vs. 47.52% ± 8.28%); and the percentage of necrotic sperm increased (33.27% ± 7.83% vs. 44.85% ± 9.16%) (P < .05). Finally, the oxidative insult increased the population with low mitochondrial membrane potential in these wild horses. We concluded that the high level of inbreeding of this particular breed apparently leads to a higher susceptibility to lipid peroxidation.     

Effect of Different Extenders and Seminal Plasma on the Susceptibility of Equine Spermatozoa to Lipid Peroxidation After Single-Layer Centrifugation, Through Androcoll-E

This study was conducted in an attempt to see whether single-layer centrifugation (SLC) increases the susceptibility of stallion spermatozoa to lipid peroxidation (LPO), in different extenders after removing all seminal plasma (SP). The susceptibility of stallion spermatozoa to LPO was studied before and after SLC. Each ejaculate was split, and aliquots extended with one of the three different extenders: INRA 96, Kenney's, or Equipro, and stored for 24 hours at 5°C (i). From the extended samples, an aliquot was kept as a control and the other was subjected to SLC through Androcoll-E. The selected spermatozoa were re-suspended in the appropriate extenders, without (ii) or with (iii) addition of 50% (v/v) pooled homologous SP for 24 hours at 5°C. Using ferrous sulfate as pro-oxidant, the susceptibility for LPO was flow-cytometrically assessed using the probe Bodipy^581/591-C₁₁. Sperm motility, monitored with a Qualisperm motility analyzer, increased after SLC treatment (P < .001). No significant correlations were found between motility and induced LPO with ferrous sulfate. The SP and extenders, per se, did not have a significant protective effect against LPO, but the interaction between SP and Kenney increased the susceptibility to LPO. However, the selected spermatozoa through Androcoll-E and the subsequent dilution in INRA had a significant protective effect against LPO (P < .05), especially when the oxidative insults were higher (80 μM).     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

鸡蛋中的脂类过氧化反应与保护

鸡蛋是人的重要食物,但其在贮藏、烹制和加工过程中发生的过氧化作用对鸡蛋的稳定性和质量影响较大。脂类的过氧化作用是自由基对不饱和脂肪酸的作用,过氧化反应发生的过程,也是一个导致脂肪酸酸败和腐败的过程。贮藏的时间、温度、光照和制作过程中的ｐＨ值等均能影响鸡蛋中脂质过氧化物。合成抗氧化剂已广泛应用于食品行业,但为安全起见,人们正在积极研究天然抗氧化剂,特别是从植物中提取的天然抗氧化剂。１　鸡蛋中的脂类过氧化反应及其机理人们早就在鸡蛋及其制品中发现了脂质过氧化物（ＬＰＯ）（Ｔｓａｉ,１９８５;Ｐｉｋｅ,…     

Relation Among Neutrophil Enzyme Activity,Lipid Peroxidation,and Acute-Phase Response in Foal Heat in Mares

Apart from functional abnormalities, genetic structural disorders and management problems endometritis is one of the major causes of infertility or subfertility in mares. However, the causes of postbreeding endometritis in foal heat have not been clearly resolved to date. The aim of this study was to search for the relationship between neutrophil activity, acute-phase proteins, and oxidative status to indicate the parameters, which can influence fertility in cold-blooded mares in foal heat. The blood for the experiment was collected from 16 cold-blooded mares at five time points: 6–8 days before parturition, 24 hours after parturition, at the first postpartum breeding on the ninth day, 24 hours after breeding, and 48 hours after ovulation. The obtained samples were assigned for hematological tests, assays of neutrophil activity, plasma malondialdehyde (MDA), and fibrinogen concentrations. We estimated that in susceptible mares during persistent postbreeding endometritis, neutrophil activity increased together with MDA and fibrinogen plasma level. Elastase release in resistant mares before parturition was 48.91 ± 1.75%, whereas in susceptible animals, the value reached 45.57 ± 1.9% of the maximal release. Myeloperoxidase release in resistant mares before parturition reached 13.95 ± 2.1%, then increased at three consecutive measurements, and returned to a value from before parturition at the last measurement. Myeloperoxidase level in susceptible mares was slightly lower than in resistant ones, then these values augmented at all measurements, reaching the maximum at the fourth one. The obtained results may help to indicate the predisposition to persistent postbreeding endometritis in cold-blooded mares bred at foal heat.     

Colloidal Centrifugation of Stallion Semen Results in a Reduced Rate of Sperm DNA Fragmentation

Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight‐line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37°C for 24 h to determine the rate of SDF (r‐SDF), a lower r‐SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ± 0.4%/h) or no centrifugation (r‐SDF = 1.2 ± 0.3%/h). These results confirm that colloidal separation of stallion spermatozoa results in prolonged sperm DNA longevity, but these differences were only apparent following a period of incubation and dynamic assessment. Consequently, we strongly recommend the use of the dynamic form of the SDF assay for evaluating centrifugation and/or other ex vivo procedures, as a single basal assessment of SDF may inadvertently result in a false‐positive evaluation of DNA quality.     

Assessment of Sperm DNA Fragmentation in Stallion (Equus caballus) and Donkey (Equus asinus) Using the Sperm Chromatin Dispersion Test

Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.     

Characterization and Localization of Membrane Vesicles in Ejaculate Fractions from the Ram, Boar and Stallion

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (+/- s.e.m.) vesicle diameters were 130.9 +/- 3.22 (range 18-577), 164.1 +/- 4.42 (range 15-671) and 159.7 +/- 2.92 nm (range 22-986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi-laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule-dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.     

Stallion Sperm Integrity After Centrifugation to Reduce Seminal Plasma Concentration and Cool Storage for 4 days

The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 10⁶ sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator for the remainder of the cooling period. At day 0, 2, and 4, total motility (TM), progressive motility (PM), and plasma (PLM) and acrosomal membrane integrity were assessed. Centrifuged groups had higher TM and PM at day 4 than the control group (P < .05). Likewise, centrifuged groups had higher intact PLM in day 4 (P < .05). A single centrifugation cycle to reduce seminal plasma concentration will suffice to preserve sperm integrity acceptable for an artificial insemination dose up to 4 days of cool storage.     

Motility and Plasma Membrane Integrity of Spermatozoa in Fractionated Stallion Ejaculates after Storage

With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with extender (Expt 1) or frozen in liquid nitrogen (Expt 2). In Expt 1, cup 1 was pre-sperm fluid, cups 2 and 3 sperm-rich fractions, and cup 4 sperm-poor fractions. In Expt 2, cups 1 and 2 were sperm-rich fractions, and cups 3 and 4 sperm-poor fractions. One sample (WE) represented the whole ejaculate in both experiments. Motility parameters were determined with a Hamilton-Thorn Motility Analyzer, and plasma membrane integrity was assessed using carboxyfluorescein diacetate and propidium iodide staining and fluorescence microscopy. The removal of seminal plasma lowered motility values, but not plasma membrane integrity, in both experiments. No significant differences between cups were observed after cooled storage. The cups differed significantly in most post-thaw motility parameters, and the sperm-rich fraction showed higher post-thaw motility than the whole ejaculate.     

Sperm Distribution and Its Relation to Ovulation in Artificially Inseminated Heifers

Contents: Blood samples were taken in ten heifers every two hours during prooestrus and oestrus for determination of the LH peak, which was used to calculote the time of expected ovulation. Artificial insemination with frozen/thawed semen was performed at different times from the beginning of oestrus and the animals were slaughtered two, twelve or 24 hours after insemination. The genital tract was flushed and the spermatozoa recovered from the flushing solution by microscopic investigation. Heifers slaughtered two hours after insemination and more than 19 hours before ovulation had more spermatozoa in the uterotubal junction (UTJ) than in the isthmus and ampulla. This sperm distribution differed from that in the other animals. In animals which were slaughtered closer to ovulation and in those who had already ovulated at slaughter, approximately the same number of spermatozoa were recovered from the UTJ and isthmus and only a few spermatozoa were recovered from the ampulla. Motile spermatozoa were found in both the isthmus, UTJ and uterus at two and twelve hours after insemination and approximately ten hours before ovulation. Their number decreased with time but some motile spermatozoa were also recovered 24 hours after insemination and after ovulation. The distribution of spermatozoa in the oviducts ipsi-and contralateral to the corpus haemorrhagicum-bearing ovary was compared in heifers that had ovulated. There were no differences in the number of oviductal segments containing spermatozoa, nor were there any differences within animal in the number of spermatozoa in the oviducts. It was concluded that ovulation does not seem to cause great changes in the distribution of spermatozoa in the oviducts. Inhalt: Spermienverteilung und deren Beziehung zur Ovulation bei kunstlich besamten Jungrindern Von 10 Färsen wurden wahrend des Diöstrus und Östrus Blutproben im Abstand von 2 Std. entnommen, um den LH-Gipfel festzustellen. Dieser wurde jür die Bestimmung des erwarteten Ovulationstermins herangezogen. Inseminationen mit Gefriersperma wurden von Brunstbeginn an in verschiedenen Intervallen vorgenommen. Die Tiere wurden 2, 12 oder 24 Std. nach der Besamung getötet, die Geschlechtsorgane gespült und die Spermien in den Spülflüssigkeiten mikroskopisch nachgewiesen. Färsen, die 2 Std. nach der Besamung und mehr als 19 Std. vor der Ovulation getötet wurden, hat-ten mehr Spermien im Ostium uterinum des Eileiters (OU) als im Isthmus und Ampulle. Diese Verteilung war von jener der anderen Tiere verschieden. Tiere, die kürzere Zeit vor der Ovulation getötet wurden, oder bei der Schlachtung schon ovuliert hatten, zeigten etwa dieselbe Anzahl von Spermien in OU und Isthmus, in der Ampulle wurden nur wenig Spermien gefunden. Bei Färsen, die ovuliert hatten, wurde die Sper-mienverteilung in den ipsi- und contralateralen Eileitern zum Corpus haemowhagicum des Ovars verglichen. Die Spermienxahlen in den Eileitersegmenten waren nicht verschieden und auch im rechten und linken Eileiter gleich. Es wurde gefolgert, daβ die Ovulation keinen groβen Einfluβ auf die Spermienverteilung in den Eileitern bewirkt.     

Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics

In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen–thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen–thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1–S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1–FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1–S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.