Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants

OBJECTIVE: To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. SAMPLE POPULATION: 2 ejaculates collected from each of 5 stallions. PROCEDURE: Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry. RESULTS: The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for alpha-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa. CONCLUSIONS AND CLINICAL RELEVANCE: The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.     

Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.     

Breeding soundness examination of North American bison bulls

OBJECTIVE: To evaluate the breeding soundness examination procedure in plains bison bulls. DESIGN: Multiyear (1993 through 1997) cross-sectional clinical procedure evaluation. ANIMALS: Two hundred thirty-four 28- to 30-month-old bison bulls at Custer State Park. PROCEDURE: Breeding soundness examinations were performed on all bison bulls using 1992 Society for Theriogenology guidelines for beef cattle semen evaluation and reproductive tract examination. Linear and logistic regression analyses were used to detect correlations and associations among breeding soundness examination variables. RESULTS: Scrotal circumference (SC) was significantly correlated with body weight, percentage of normal spermatozoa, percentage of primary spermatozoal defects, and percentage of motile spermatozoa. Scrotal circumference was positively associated with increased odds of semen collection, satisfactory motility (> or = 30% motility), satisfactory morphology (> or = 70% normal spermatozoa), and simultaneous satisfactory motility and morphology. Receiver-operator characteristic curve analysis selected 29 cm as the optimal SC cutoff most predictive of simultaneous satisfactory spermatozoal motility and morphology. Only 36.2% (83/229) of the bison bulls had a SC of 29 cm or greater and satisfactory spermatozoal motility and morphology. CLINICAL IMPLICATIONS: SC is a good indicator of adequate spermatozoal motility and structure in bison. We recommend use of 30% spermatozoal motility, 70% normal spermatozoal morphology, and 29-cm SC as minimal satisfactory measurements for breeding soundness examinations of 28- to 30-month-old bison bulls that have been raised on forage-based nutrition.     

影响马精液冷冻保存效率的因素分析

马精液冷冻保存技术对提高马的繁殖效率有重要意义,多种因素会对冷冻保存精液的精子造成损害。本文综述了精清、抗冻剂、微生物、过氧化作用、渗透压、冷冻和解冻等影响马精子冷冻保存的因素,旨在为提高马精液冷冻保存技术的效率、促进我国马产业更快发展提供理论依据。     

The effect of cryopreservation on the capacitation status and epithelial cell attachment capability of dog spermatozoa

Freezing and cooling of spermatozoa during cryopreservation for artificial insemination causes ultrastructural changes in the acrosome and plasma membrane which reduces longevity and fertility. Cryopreservation-induced capacitation-like changes and reduced ability of spermatozoa to bind to the cells of the reproductive tract of the bitch may contribute to the reduced fertility of cryopreserved spermatozoa. Previous studies in the dog have investigated the effects of extending and cooling spermatozoa on the plasma membrane but often only after freeze-thawing and not in conjunction with an assessment of their ability to bind to uterine tube epithelial explants. This study investigated the effect of each stage of the cryopreservation process on capacitation and attachment to the reproductive tract of the bitch. The capacitation status of spermatozoa was studied over time after cryopreservation using a chlortetracycline and Hoechst 33258 stain. The ability of spermatozoa to bind to uterine tube epithelial explants was studied using Hoechst 33342 stain. Extending, cooling and freeze-thawing promoted capacitation and decreased the spermatozoal binding ability. The effect of each stage appeared to be cumulative with the freeze-thawing stage being the most dramatic. The results suggested that the cumulative effect of each stage of the cryopreservation process results in the promotion of capacitation before spermatozoa have reached the site of fertilisation, and therefore spermatozoa have reduced ability to attach to epithelial cells. These effects are contributory factors to the reduced fertility of cryopreserved spermatozoa.     

Association between Myeloperoxidase Concentration in Equine Frozen Semen and Post‐Thawing Parameters

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro‐oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty‐five straws from different stallions were analysed. Post‐thawing spermatozoal concentration, and progressive and total motility were determined by Computer‐Assisted Semen Analysis. Freezability was determined according to post‐thawing progressive motility (above or below 15%). Percentage of alive spermatozoa and abnormal forms was determined after Eosin–Nigrosin and Diff‐Quick^® staining, respectively. Post‐thawing MPO concentration was measured by enzyme‐linked immunosorbent assay (ELISA). Our study shows that frozen thawed semen contains large amounts of free MPO. We also observed that post‐thawing MPO ELISA assay can be used as an indicator of equine semen freezability. High MPO concentration samples showed lower total and progressive motility. A higher proportion of abnormal head shape associated with acrosome reaction was observed in our late examinations of the high concentration MPO group. Our results show that MPO adversely affects total and progressive motility of equine semen. A negative correlation between normal motile forms and MPO concentration was also observed. The effect of MPO on dead or abnormal forms remains to be precised.     

Use of a computerized system for evaluation of equine spermatozoal motility

Three ejaculates from each of 3 stallions were used to evaluate a computerized system (Hamilton-Thorn motility analyzer; HTMA) for measuring equine spermatozoal motility. Variance components (ejaculate-within-stallion, chamber-within-ejaculate, and microscopic field-within-chamber) were determined for each stallion after diluting ejaculates to 25 x 10(6) spermatozoa/ml with a skim milk-glucose seminal extender. The HTMA was compared with frame-by-frame playback videomicrography (VIDEO) for determining: percentage of spermatozoal motility and spermatozoal number in microscopic fields; curvilinear velocity and straight-line velocity of individual spermatozoa for 5 track types; and repeatability of those velocity measurements. The effect of spermatozoal number per microscopic field on incidence of intersecting spermatozoa and the outcome of intersecting spermatozoa also were evaluated. Greatest variability in motility measures was generally attributed to the microscopic field-within-chamber component. The HTMA was highly correlated with VIDEO for estimation of spermatozoal numbers per microscopic field (r = 0.99; P less than 0.001) and motility (r = 0.97; P less than 0.001); however over the entire range of spermatozoal numbers, the HTMA yielded higher spermatozoal numbers per microscopic field (P less than 0.05) and higher motility (P less than 0.05) than did VIDEO. The HTMA- and VIDEO-derived measurements of curvilinear and straight-line velocities were highly correlated for all spermatozoal track types, but both measures were higher (P less than 0.05) by use of the HTMA than by use of VIDEO for most track types. For 3 of 5 track types, measurements of curvilinear and straight-line velocities were less variable (P less than 0.05), using the HTMA, rather than VIDEO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

Physiological changes in fowl and turkey spermatozoa during in vitro storage

1. Although early work on semen storage has been rather empirical in approach, only basic research can provide a framework of biological mechanisms from which improvements in the techniques of cryopreservation and liquid semen storage can progress logically. 2. A major drawback in this work has been the lack of adequate tests for quantitating and differentiating aspects of 'fertility'. 3. Basic research has now provided techniques for assessing: sperm fertilising ability in terms of numbers of fertile eggs; the efficiency of hens' oviducts at accepting and retaining spermatozoa, and sperm 'quality' as motility, metabolism and plasma membrane patency. 4. These techniques may be used for a more critical assessment of the effects of both cryopreservation and liquid semen storage on sperm function, although the integrity of sperm surface proteins may be a more sensitive variable which has yet to be measured. 5. Further improvements in sperm cryopreservation technology are best approached through an understanding of the fundamental cellular and molecular changes which take place during freezing; thus far little is known of such changes in avian spermatozoa. 6. The ideal milieu for maintaining spermatozoa in liquid semen storage should mimic the environment of the oviducal sperm storage tubules; elucidation of the factors involved in progressing steadily.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

Proteomic identification of cryostress in epididymal spermatozoa

Background: Cryopreservation of epididymal spermatozoa is important in cases in which it is not possible to collect semen using normal methods, as the sudden death of an animal or a catastrophic injury. However, the freezing and thawing processes cause stress to spermatozoa, including cold shock, osmotic damage, and ice crystal formation,thereby reducing sperm quality. We assessed the motility(%), motion kinematics, capacitation status, and viability of spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining.Moreover, we identified proteins associated with cryostress using a proteomic approach and performed western blotting to validate two-dimensional electrophoresis(2-DE) results using two commercial antibodies.Results: Cryopreservation reduced viability(%), motility(%), straight-line velocity(VSL), average path velocity(VAP), amplitude of lateral head displacement(ALH), and capacitated spermatozoa, whereas straightness(STR)and the acrosome reaction increased after cryopreservation(P 0.05). Nine proteins were differentially expressed(two proteins decreased and seven increased)(3 fold, P 0.05) before and after cryopreservation. The proteins differentially expressed following cryopreservation are putatively related to several signaling pathways, including the ephrinR-actin pathway, the ROS metabolism pathway, actin cytoskeleton assembly, actin cytoskeleton regulation,and the guanylate cyclase pathway.Conclusion: The results of the current study provide information on epididymal sperm proteome dynamics and possible protein markers of cryo-stress during cryopreservation. This information will further the basic understanding of cryopreservation and aid future studies aiming to identify the mechanism of cryostress responses.     

Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa

Two experiments were conducted to test whether stallionand/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, 15 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/ml) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment 1, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.     

马精液冷冻保存技术研究进展

马精液冷冻保存是一种将种公马精液加入抗冻保护剂,再进行一系列处理,最后保存在超低温中(液氮、干冰)的技术。近年来该技术随着马冷冻精液在许多国家马匹繁殖育种中逐渐应用而得到了较好的发展。作者综述了马精液冷冻保存技术的精液稀释、离心、再稀释、降温平衡、分装剂型、降温冷冻、液氮保存的研究进展,旨在对马精液冷冻保存技术的进一步研究提供一定的参考,以期推动中国现代马业的健康可持续发展。     

Commercial Breeding with Sexed Stallion Semen: Reality or Fiction?

Sorting stallion spermatozoa for the purpose of separating X- and Y-bearing spermatozoal populations has had significant advances in the past 10 years. However, current technology requires that the mare be close to the laboratory processing the semen. The development of procedures to cool and freeze sex-sorted sperm will be critical if this technology is to be embraced by the equine industry worldwide.     

Assessment of fresh extended equine semen samples using spectrophotometry and resazurin

The purpose of this study was to determine if spectrophotometric assessment of resazurin dye in fresh extended equine semen samples was associated with spermatozoal parameters.This technique could be beneficial to veterinarians and horse producers for evaluating semen samples prior to artificially inseminating a mare. The reducible dye resazurin (blue color) is reduced via an oxidation-reduction reaction in the presence of metabolically active spermatozoa to resorufin (pink color), and upon further reduction to dihydroresorufin (colorless). Sixty semen samples were collected from six stallions (5 Quarter Horse and 1 Arabian) using a Missouri style artificial vagina. Sample aliquots were diluted using a 1:30 (semen: extender) ratio with a non-fat dry skim milk (NFDSM) glucose extender T. The diluted sample was then assessed microscopically at 250x to determine concentration, the number of motile, and progressively motile spermatozoa/mL. The remainder of the sample was diluted at a 1:1 (semen: extender) ratio prior to dye incubation and spectrophotometric analysis. The resazurin dye (50 μL from a 0.338 mM solution) was added to 4 (2 mL) aliquots of extended sample, thoroughly mixed, and incubated at 37°C. Butyl alcohol (4.8 mL) was added at five-minute increments (0,5, 10, and 15 minutes) to stop spermatozoal metabolism and draw the color out of the sample. Each aliquot was then vortexed prior to centrifugation at 700xg to extract the butanol color layer. Spectrophotometric absorbance values (615 nm) of the butanol color layer were recorded. Relationships between spectrophotometric absorbance values and spermatozoal parameters were assessed using correlation analyses on square root transformed data. At the 0 minute incubation time there were no associations between spermatozoal parameters and spectrophotometric absorbance values. However, at the five minute incubation time the spectrophotometric absorbance values were negatively correlated with concentration (r=−0.31; P=0.02), number of motile (r=−0.27; P=0.04) and progressively motile (r=−0.30; P=0.02) spermatozoa/mL. At the 10 minute incubation time negative correlations were observed between the spectrophotometric absorbance values and concentration (r=−0.48; P=0.0001), number of motile (r=−0.45; P=0.0004) and progressively motile (r=−0.46; P=0.0002) spermatozoa/mL. At the 15 minute incubation time negative correlations were also found between spectrophotometric absorbance values and concentration (r=−0.52; P=0.0001), number of motile (r=−0.50; P=0.0001) and progressively motile (r=−0.52; P=0.0001) spermatozoa/mL. Spectrophotometric absorbance values were associated with spermatozoal parameters at the 5, 10, and 15 minute incubation times.     

Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa

OBJECTIVE: To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. PROCEDURE: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. RESULTS: Following centrifugation, spermatozoa diluted with modified Kenney's Centrifugation Medium (MKCM) displayed a higher percentage of (normal) F pattern (94.3%) compared with spermatozoa in Kenney's Centrifugation Medium (KCM) (84.9%) and Glucose-EDTA Centrifugation Medium (GECM) (85.2%). Conversely, the percentage of spermatozoa displaying the (capacitated) B pattern was higher in the KCM (14.1%) and GECM (13.8%) than in the MKCM (5.0%). Following freezing-thawing, there were lower percentages of spermatozoa displaying the AR (acrosome reacted) pattern in modified Kenney's Freezing Medium (MKFM) (45.6%) compared with Kenney's Freezing Medium (KFM) (61.4%) and lactose-EDTA Freezing Medium (LEFM) (61.1%). There was a correspondingly higher percentage of spermatozoa displaying the B pattern in MKFM (52.3%) compared with KFM (37.9%) and LEFM (38.6%). There was no significant difference between the freezing media in the percentage of spermatozoa displaying the F pattern. The percentage of progressively motile spermatozoa was also influenced by the type of freezing medium (P < 0.001). Post-thaw percentages of progressively motile spermatozoa, frozen in MKFM, KFM, and LEFM, were 31.4, 25.8 and 23.3%, respectively. CONCLUSION: MKFM was the preferred medium for cryopreservation of equine spermatozoa due to its superior protection against changes in motility and membrane quality compared with the other freezing media studied.     

Effects of Pentoxifylline,Caffeine, and Taurine on Post-Thaw Motility and Longevity of Equine Frozen Semen

Frozen semen provides several advantages to the breeder relative to fresh or cooled semen. However, some stallions are undesirable candidates for semen freezing due to poor post-thaw motility or longevity caused by membrane damage, osmotic stress, and oxidative stress during cryopreservation. The objective of this study was to determine the effect of post-thaw addition of pentoxifylline, caffeine, or taurine on sperm motility and longevity in equine frozen semen. Pentoxifylline, caffeine, or taurine was incorporated immediately into thawing frozen semen from nine warmblood stallions. Spermatozoa motility and longevity parameters were recorded and analyzed for each additive and for an untreated control. Of the three additives, only pentoxifylline improved total and progressive semen motility relative to that of untreated control. Pentoxifylline also increased semen curvilinear velocity, average path velocity, and straight line velocity relative to those of caffeine, taurine, or control. Semen treated with pentoxifylline also showed greater longevity relative to that of caffeine- or taurine-treated or untreated semen. Taurine improved linearity in comparison to that of semen treated with pentoxifylline, caffeine, or control but did not improve other parameters. Pentoxifylline may be useful in enhancing the quality of equine frozen semen and therefore improving its fertility. Additional studies are warranted that examine the effect of these additives on the conception rate. Pentoxifylline can be used to increase motility and longevity of equine frozen semen and theoretically increase probability of conception and overall breeding success rates.     

Effect of method of collection on seminal characteristics of the domestic cat

The effect of level of voltage and method of collection on seminal characteristics were studied in the domestic cat. A Latin square design was used to determine the effects of voltage (1, 2, 4, or 6 V) on seminal characteristics of electroejaculates for 8 cats (experiment 1). There was a significant effect of cat on the total volume (P less than 0.005), number of spermatozoa (P less than 0.05), pH (P less than 0.05), and osmolality (P less than 0.025). There was an effect of week for the pH (P less than 0.05) and osmolality (P less than 0.005) of semen. Voltage of stimulation affected the total volume (P less than 0.005), total number of spermatozoa (P less than 0.025), and osmolality (P less than 0.005) of semen. There were trends (P less than 0.1) for an effect of cat and an effect of voltage on spermatozoal motility. Urine contamination was not observed in any of the electroejaculates. A 2, 2 X 2 Latin square design was used in experiment 2 to determine the effect of method of collection (artificial vagina or electroejaculation) on seminal characteristics for 4 cats. Electroejaculation was performed, using the 6-V electrical stimulus selected from experiment 1. There was a significant (P less than 0.025) effect of cat on the motility of the spermatozoa in the viability preparation and a trend (P less than 0.1) for an effect of cat on spermatozoal viability.(ABSTRACT TRUNCATED AT 250 WORDS)