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1.
Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.  相似文献   

2.
The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 106/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 106 ± 21, 59 × 106 ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.  相似文献   

3.
Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode's medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.  相似文献   

4.
Colloid centrifugation of boar semen has been reported sporadically for at least the last two decades, beginning with density gradient centrifugation (DGC) and progressing more recently to single layer centrifugation (SLC). Single layer centrifugation through a species-specific colloid has been shown to be effective in selecting the best spermatozoa (spermatozoa with good motility and normal morphology) from boar sperm samples. The method is easier to use and less time-consuming than DGC and has been scaled-up to allow whole ejaculates from other species, e.g. stallions, to be processed in a practical manner. The SLC technique is described, and various scale-up versions are presented. The potential applications for SLC in boar semen preservation are as follows: to improve sperm quality in artificial insemination (AI) doses for 'problem' boars; to increase the shelf-life of normal stored sperm samples, either by processing the fresh semen before preparing AI doses or by processing the stored semen dose to extract the best spermatozoa; to remove pathogens (viruses, bacteria), thus improving biosecurity of semen doses and potentially reducing the use of antibiotics; to improve cryosurvival by removing dead and dying spermatozoa prior to cryopreservation; to select spermatozoa for in vitro fertilization. These applications are discussed and practical examples are provided. Finally, a few thoughts about the economic value of the technique to the boar semen industry are presented.  相似文献   

5.
The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.  相似文献   

6.
The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.  相似文献   

7.
Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106 sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm®. A single layer of 40% PureSperm® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.  相似文献   

8.
The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 106 spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.  相似文献   

9.
Although several selection techniques are available for processing spermatozoa, only colloid centrifugation has been used to any extent in this field, starting with density gradient centrifugation and progressing more recently to single-layer centrifugation (SLC). SLC through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility and normal morphology from stallion semen. The method is easier to use and less time-consuming than density gradient centrifugation, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The potential applications of SLC in equine breeding are as follows: to improve sperm quality in artificial insemination doses for “problem” ejaculates, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding.  相似文献   

10.
Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two‐layer iodixanol as well as single‐layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen collection with centrifugation after 1‐day refrigerated storage. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that both two‐ and single‐layer density centrifugation processing resulted in higher percentages of morphologically normal and motile sperm with higher membrane and chromatin integrity, as compared to standard centrifugation or diluted samples. Differences were only in the order of magnitude of 5%. Recovery rates after density centrifugation were only approximately 30–40%. When cryopreservation was carried out after 1‐day refrigerated storage, centrifugation processing of sperm directly after semen collection resulted in higher percentages of plasma membrane intact sperm post‐thaw as compared to performing centrifugation processing of stored sperm just prior to cryopreservation. No significant differences in progressively motile sperm post‐thaw were seen. Taken together, for delayed cryopreservation, it is best to perform density centrifugation directly after collection rather than immediately prior to cryopreservation.  相似文献   

11.
Insemination with chilled transported semen has become distinctly important in the horse-breeding industry. To ensure cell survival during cooled storage, semen is diluted with an appropriate extender and the concentration of seminal plasma (SP) is reduced. Nevertheless, SP plays an important immunomodulatory role in the female genital tract and supports sperm fertility. The aim of the present study was to evaluate the effect of the addition of autologous SP after cooled storage to highly concentrated stallion semen. Therefore, SP was removed by simple centrifugation of extended semen, aspiration of the supernatant, and resuspension of the sperm pellet with semen extender. Motion characteristics were evaluated after cooled storage for 48 hours at concentrations of 333 × 106 sperm/mL in comparison with stored samples at concentration of 25 × 106 sperm/mL (control). The highly concentrated semen samples were diluted with an extender containing 0%, 5%, 20%, and 80% SP directly before motility analysis. Dilution of the cooled semen with a fresh semen extender without SP (0%) increased kinematic parameters (curvilinear velocity [VCL] 137.3 vs. 151.8; straight-line velocity [VSL] 49.0 vs. 57.5; average path velocity [VAP] 69.5 vs. 79.4 μm/second; amplitude of lateral head [ALH] 3.1 vs. 3.3 μm; beat cross frequency [BCF] 31.6 vs. 33.5 Hz; P < .05) but not total motility (51% vs. 43%) and progressive motility (46% vs. 36%) compared with controls. The addition of SP after storage for 48 hours decreased sperm total motility and progressive motility regardless of SP concentration: 5 (38% and 34%), 20 (37% and 33%), and 80% SP (27% and 22%; P < .05). In contrast, kinematic parameters were enhanced by extenders containing 5% and 20% SP (VCL: 148.0 and 155.6; VSL: 59.2 and 60.9; VAP: 78.7 and 81.9; BCF: 33.4 and 35.7; ALH: 3.4 and 3.4; P < .05). However, using an extender containing 80% SP was detrimental to kinematic parameters (VCL: 151.2; VSL: 52.2; VAP: 76.9; BCF: 34.8; P < .05) except for ALH, which increased (3.5; P < .05). In conclusion, cooled storage at concentrations of 333 × 106 sperm/mL did not affect sperm motility. The addition of a fresh extender or an extender containing small concentrations of SP to highly concentrated ejaculated sperm increased kinematic values after storage; however, increasing concentrations of SP decreased sperm motility.  相似文献   

12.
Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey’s test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.  相似文献   

13.
The objective of this study was to improve the quality of cryopreserved–thawed equine sperm using single-layer density centrifugation (SLC). Sperm quality was assessed by DNA integrity, motility, morphology, mitochondrial membrane potential, viability, and plasma membrane alteration. The percentage of DNA-damaged sperm (expressed in % COMP) was lower (P = .001) after SLC (1.6 ± 0.5% vs. 6.8 ± 0.5%). Total sperm motility (80 ± 2.4% vs. 41.7 ± 2.4%) and progressive sperm motility (69.5 ± 2.9% vs. 31.5 ± 2.9%) (P < .001), as well as the percentage of morphologically normal sperm (45 ± 3.9% vs. 27.7 ± 3.9%), increased after SLC compared with control sample. In addition, the proportion of sperm with high mitochondrial membrane potential increased (81.6 ± 1.8% vs. 42.1 ± 1.8%), as did the viability of sperm (71.1 ± 2.4% vs. 39.5 ± 2.4%), after SLC compared with the control sperm. The proportion of sperm with alteration in plasma membrane structure was lower after SLC compared with control sample (6.4 ± 1.1% vs. 18.9 ± 1.1%). Overall, sperm recovery was 72.7 ± 3.6% in the control sample compared with 14.8 ± 3.6% after SLC (P < .001). We conclude that based on the sperm parameters evaluated, SLC improves the quality of cryopreserved–thawed equine spermatozoa.  相似文献   

14.
ABSTRACT

  • 1. This study examined different glycerol concentrations (GC) and freezing rates to improve the quality of rooster spermatozoa frozen in straws, and to determine the effect of varying GC on post-thawed spermatozoa quality, as evaluated by fertility and hatchability.

  • 2.The experiment included two tests. In test 1, rooster semen straws containing 2, 4, 6, 8 and 11% glycerol were put in a rack (nine tiers with a 1 cm interval between every two tiers, 1 to 9 cm above liquid nitrogen (LN) source), and gradually frozen. The semen straws located in different tiers experienced different temperatures and freezing rates. The straws were then thawed and live sperm numbers determined. In test 2, rooster semen straws containing 2, 4, 6, 8 and 11% glycerol were put on optimal tiers (identified in test 1) for freezing, and stored at ?196°C. Hens were inseminated with the frozen semen (post-thawed and glycerol removed, about 4.0 × 108 sperm per hen), and eggs incubated.

  • 3. The numbers of live sperm in the 11% glycerol group was higher than that in 2, 4 or 6% glycerol group (P < 0.05) for the semen straws on tiers 1 to 9, while that on tiers 1 to 5 was lower than that on tier 6 to 8 (P < 0.05). GC, freezing rate and the interaction between GC and freezing rate had a significant effect on live sperm numbers (P < 0.01). The highest fertility was in the 6% glycerol group and occurred on day 5 after insemination. The lowest fertility occurred in the 2% glycerol group on day 10 after insemination.

  • 4. The optimal combination was 11% glycerol in straws located 6 cm above the LN surface (on tier 6). The 6% glycerol group achieved the highest fertility (77.6%), which surpassed that reported in recent years.

  相似文献   

15.
In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca2+-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca2+]i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca2+-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca2+-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.  相似文献   

16.
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.  相似文献   

17.
Influences of seminal plasma and extender on sperm motility, ATP-concen-tration, and the activity of acid and alkaline phosphatases of beagle dog semen. The percentage of progressively motile spermatozoa, the time of sperm survival, ATP-concentration, and the activity of the acid and alkaline phosphatases were determined in the semen of five healthy beagle dogs immediately after collection and after storage for 24 hours at +5°C. The sperm rich fraction was examined in the native state as well as after the addition of prostatic secretion and a Tris-eggyolk-medium, resp. The percentage of progressively motile spermatozoa was 64.4% in the fresh undiluted semen, 68.4% after the addition of prostatic secretion, and 74.8% after dilution with Tris-eggyolk-medium. It decreased within 24 hours to 31.6%, 20.4%, and 59.2%, resp. After 0 und 24 hours, resp., the sperm survival time in a coverslide preparation was  相似文献   

18.
This study evaluated measures of sperm quality in relation to fertility achieved with fresh semen or semen cooled and stored. Semen from 1 stallion was collected and processed to provide 3 treatments: group 1 received fresh semen; group 2 received cooled semen containing 50% seminal plasma (SP) stored for 4 days; and group 3 received cooled semen containing 50% SP stored for 1 day, then centrifuged and resuspended in fresh extender containing 10% SP on days 1 to 3. Inseminates were evaluated for sperm motion characteristics and the percentage of sperm with intact membranes (SMI). Mares (n = 34) in estrus were treated with an ovulation-inducing drug and inseminated with 100 million membrane-intact sperm on the following day. Pregnancy status was determined via transrectal ultrasonography 2 weeks after ovulation. The mean percentage of SMI was higher in group 1 (81%, initial) than in group 2 (74%, day 4) or group 3 (74%, day 4) (P < .05). The median percentages of total sperm motility differed among the groups (77%, 5%, 59% for groups 1, 2, and 3 respectively; P < .05). Median values for the percentages of progressively motile sperm and curvilinear velocity for group 1 (55%, 216 μm/s) and 3 (37%, 186 μm/s) were higher than for group 2 (1%, 73 μm/s) (P < .05). Pregnancy rates did not differ among groups (5 of 11, 45% in group 1; 5 of 11, 45% in group 2; and 7 of 12, 58%, in group 3; P = .77). These data suggest that, at least for this stallion, sperm membrane integrity may be a more valuable means of assessing potential fertility of cooled-stored semen than sperm motion characteristics.  相似文献   

19.
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.  相似文献   

20.
Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single‐layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll‐E, a species‐specific colloid were evaluated for their ability to separate spermatozoa from virus in ejaculates from carrier stallions. The three types of SLC significantly reduced the virus titre in fresh semen at 0 h and in stored semen at 24 h (p < 0.001) but did not completely eliminate the virus. Sperm motility parameters such as total motility and progressive motility were significantly increased after colloid centrifugation, whereas curvilinear velocity and amplitude of lateral head deviation were decreased, and the remainder (straight line velocity, average path velocity, straightness, linearity, wobble and beat cross‐frequency) were not significantly affected by the processing. Although virus titres were reduced in the SLC samples, significant levels of infectivity still remained, especially in stallions shedding large amounts of virus. It remains to be determined whether SLC‐processed sperm samples from stallions shedding low virus titres retain sufficient equine arteritis virus to cause infection in mares through artificial insemination.  相似文献   

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