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Effects of Dead Sperm on Motion Characteristics of Live Sperm in
Fresh and Cooled-Stored Equine Semen Introduction Cooled-stored transported equine semen has been in widespread use for almost twenty years. The current industry standard for cooled-stored semen is to dilute semen in a NFDMS-Gluc extender containing antibiotics, to a final sperm concentration of 25 - 50 x 106 sperm/mL. However, some workers base this concentration on progressively motile sperm (PMS) while others use total sperm. For stallions with marginal progressive motility in their fresh ejaculates, packaging the semen based on PMS/mL could result in samples containing large numbers of dead or dying sperm. The aim of this study was to determine if dead sperm adversely affected motion characteristics of live sperm in cooled-stored equine semen. Materials and Methods Three ejaculates from each of 3 stallions were centrifuged and all seminal plasma was removed. Sperm were resuspended to 25 x 106 sperm/mL with EZ-Mixin CST extender and 10% autogenous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50 or 75% (v/v) dead sperm were added from pre-prepared sperm suspensions. Dead sperm preparations contained 25 x 106 sperm/mL and 10% seminal plasma from pooled ejaculates of the 3 stallions, in EZ-Mixin CST extender. Sperm were killed in the pooled ejaculates by repeated (3x) freezing in LN2 and thawing at 50°C. Suspensions of dead sperm were stored at -20oC until warmed to 37oC and mixed with aliquots of fresh ejaculates to be cooled and stored at in an Equitainer for 24 h. Motion characteristics (% total motility [MOT], % progressive motility [PMOT], curvilinear velocity [VCL] and linearity [LIN]) of fresh and 24 h cooled samples were determined using a Hamilton-Thorne IVOS 10 CASA system. Within ejaculates, observed MOT, and PMOT were adjusted for comparison by the following formula: adjusted value = observed value + (1 - % dead sperm added). Adjusted MOT (adjMOT) and PMOT (adjPMOT) as well as VCL and LIN for each treatment were compared in fresh and 24 h cooled-stored samples using repeated measures ANOVA. Data are presented as adjMOT and adjPMOT. Results No differences (P>0.05) were detected for adjMOT, adjPMOT or LIN among treatments within fresh and cooled-stored semen samples. Within fresh samples, VCL was lower in aliquots to which 75% dead sperm were added compared to other treatments (P<0.05). However, no differences were detected in VCL among treatment groups in 24 h cooled-stored semen samples (P>0.05). The mean (+ sem) reductions in MOT (6.4 + 1.6 %) and PMOT (8.9 + 1.1%) in control groups were minimal, and did not differ among treatment groups (P>0.05; data not shown).
Discussion and Conclusions The presence of dead sperm did not affect motion characteristics of live sperm in fresh or cooled-stored equine semen samples in this study. The stallions used in this study produced sperm that had very good tolerance to cooling and storage as evidenced by the fact that after 24 h of cooling and storage, MOT and PMOT were only reduced by 6% and 9%, respectively. In addition, MOT and PMOT in control samples did not differ between fresh and cooled-stored samples. It is possible that if stallions producing less resilient semen had been used in this study, potential adverse effects of the addition of dead sperm would have been detected. It is also possible that different results may have been obtained if higher concentrations of sperm or seminal plasma had been used. In the present study, the sperm concentration (25 x 106 total sperm/mL) and seminal plasma level (10%) were constant for all treatments. If an ejaculate containing <50% PMS is diluted and packaged at 50 x 106 PMS/mL, then this sample would contain >100 x 106 total sperm/mL. Motility of cooled-stored equine semen at these higher concentrations is reduced compared to semen packaged at 25 - 50 x 106 total sperm/mL (Varner et al. 1987). Semen processed in this manner would also contain higher levels of seminal plasma. When using NFDMS-Gluc extenders, 5 to 20% seminal plasma is considered optimal for maintaining motility after cooling and storage (Jasko et al. 1992). The combination of more concentrated sperm together with higher levels of seminal plasma, is likely to exert more deleterious effects on cooled-stored equine semen than those observed in the present study For the stallions in this study, the addition of freeze-killed dead sperm did not adversely affect motion characteristics of live sperm when equine semen containing 25 x 106 sperm/mL and 10% seminal plasma was cooled and stored for 24 h. Effects of the presence of non-viable sperm in semen samples containing higher levels of seminal plasma and more concentrated sperm warrant further study. In this study, we introduced frozen/thawed sperm into extended semen to evaluate their effects on motion characteristics of the intrinsic sperm population. It is possible that this experimental approach may not simulate the presence of necrotizing sperm (and potential products emanating from these sperm) that may adversely affect the quality of remaining viable sperm. It does suggest, however, that cryodamaged sperm do not have a detrimental impact on the remaining sperm. This observation has application to effects on sperm that are subjected to cryopreservation. References 1. Jasko DJ, Hathaway JA, Schaltenbrand WD, Simper WD, Squires EL 1992. Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 37: 1241-1252. 2. Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM 1987. Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. Theriogenology 28: 709-723. Acknowledgements Financial support for this study was obtained from the Link Equine Research Endowment Fund, Texas A&M University. |
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