The Effects of Seminal Plasma, Skim Milk, and Tyrodes Solutions on Survival of Stallion Sperm Stored at 4 °C


Abstract: This study was conducted to evaluate the effects of skim milk, skim milk extender supplemented with modified Tyrode´s medium, and seminal plasma (obtained from the sperm-rich fraction) on stallion sperm survival after 24 h cooled storage at 4 °C. Ejaculates from 3 Finnhorse stallions were used in this study. Ejaculates were collected by an automated phantom (Equidame) which separates fractions of semen into 5 cups. Only the second cup (sperm-rich fraction) was used to obtain spermatozoa and seminal plasma for the experiment. Semen was diluted with the stallion´s own second cup seminal plasma and the extenders to a final concentration of 50 × 10^6 sperm/ml. After 24-h cooled storage in Equitainer, spermatozoal motion characteristics were evaluated using a computerized motility analyzer. Spermatozoal viability was assessed by staining spermatozoa with CAM/PI (calcein AM / propidium iodide). Membrane integrity was evaluated using a fluorometer and staining with Hoechst 33258. After cooled storage, seminal plasma showed significantly lower viability and membrane integrity than the extenders and differences were significant, but total motility and progressive motility in seminal plasma and Kenney extender were not significantly different. The best total motility, progressive motility, VCL (velocity curve linear) and DCL (distance curve linear) values were observed in skim milk extender supplemented with Tyrode´s medium. The results of this study demonstrated that; 1) exposure to seminal plasma for long periods is detrimental to sperm survival, 2) skim milk glucose extender provides good protection for sperm viability and membrane integrity, but not for motility, 3) on the contrary, skim milk glucose extender supplemented with modified Tyrode´s medium is more effective in maintaining motility, presumably because of its ionic balance.

Keywords: Stallion semen, cooled storage, seminal plasma, semen extenders

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