Inhibition of Equine Spermatozoa Motility Using Sodium Tetraphenylboron and Reactivation With Caffeine-Potential to Improve Post-Thaw Recovery



Humphreys, Church

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Frozen semen provides several advantages relative to fresh or chilled semen for conservation of endangered equids. However, cryopreservation results in extensive loss of spermatozoa motility and viability arising from freeze-thaw induced membrane damage, osmotic stress, and oxidative stress leading to suboptimal fertility outcomes. The main objectives of this study were to:1) assess the ability of sodium tetraphenyl boron (TPB) to inhibit equine spermatozoa motility, 2) assess the ability of caffeine to reverse the inhibitory effects of TPB, and 3) evaluate if the inhibition of equine spermatozoa motility prior to cryopreservation improves post-thaw spermatozoa motility. Spermatozoa from domestic horse (Equus ferus caballus), Przewalski’s horse (E. f. przewalskii), and Persian onager (E. f. hemionus) were exposed to varying concentrations of TPB. Several motility characteristics including total motility, progressive motility, curvilinear motility, average path velocity, linearity, and straightness of equine spermatozoa were assessed using Computer Assisted Sperm analysis (CASA) over 2 h of in vitro incubation. After 2 h of incubation, TPB was washed and spermatozoa were resuspended in fresh medium containing 10 mM caffeine. Motility parameters described above were analyzed using CASA for 2 h of in vitro incubation. In a separate experiment, spermatozoa suspensions were pre-treated with 0 mM, 150 mM or 300 mM TPB and cryopreserved using standard protocols. Following thawing, spermatozoa were assessed for motility parameters (as described above), cell viability and acrosomal integrity. Exposure to TPB (150 mM, 300 mM and 500 mM) resulted in a significant sharp (P < 0.05) decline in total spermatozoa motility and progressive motility within 30 min of in vitro incubation. Caffeine (10 mM) failed to restore spermatozoa motility in TPB treated samples. Pre-treatment with TPB prior to cryopreservation failed to improve post-thaw sperm cell motility, progressive motility, viability or CASA parameters. However, there was no change in acrosomal integrity before or after cryopreservation. Overall, although TPB was successful in inhibiting sperm cell motility in all three subspecies of equids, contrary to earlier reports in human spermatozoa, caffeine was unable to restore motility in TPB treated spermatozoa. Furthermore, pre-treatment with TPB failed to improve post-thaw motility in equid spermatozoa. Further research is warranted to evaluate alternate cellular pathways that regulate reversible inhibition of equid spermatozoa and their effects on post-thaw sperm cell survival.


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Spermatozoa, Caffeine, Equine, Sperm cell motility, Sodium Tetraphenylboron, Cryopreservation