Comparison of three different sized SephadexⓇ beads for filtration of cryopreserved epididymal sperm

Abstract

Cryopreserving epididymal spermatozoa is often the last opportunity to preserve genetic material for stallions that suffered catastrophic injury, acute or chronic illness, and unexpected death. Epididymal sperm tend to have lower quality and pregnancy rates when compared to cryopreservation of ejaculated frozen semen. Gradient chromatography columns using Sephadex^Ⓡ beads have been used to improve the quality of stallion semen; but no data for epidydimal sperm exists. The aim of this study was to compare the efficacy of different Sephadex^Ⓡ bead sizes to improve the quality of frozen epididymal sperm. Sperm from adult stallions (n=10) was obtained by retrograde flush post-castration and frozen in a lactose-EDTA extender. The sperm were thawed and evaluated for total and progressive motility, concentration and viability (control). Sephadex^Ⓡ columns were prepared (G-25, G-50 and G-75) and sperm from each stallion divided into three equal sized aliquots, which were filtrated through each one of the columns. The filtrate was assessed for volume, sperm concentration, total and progressive motility, morphology and sperm viability. Similar percentages of sperm were recovered from all three sized Sephadex^Ⓡ beads (mean 35-37% ±19). The total motility was not significantly different between the control sperm and the Sephadex^Ⓡ column groups (mean 35% ±13; 39% ±11; 37% ±12; 36% ±16; for control, G-25, G-50 and G-75). The progressive motility of the control and Sephadex^Ⓡ groups were also similar (mean 22%±13; 28%±12; 28%±13; 28%±15; for control, G-25, G-50 and G-75, respectively). Morphological evaluations were similar between control and filtered semen for the percentage of normal sperm (mean 15% ±8; 21% ±9; 22% ±10; 25% ±9), detached heads (mean 2% ±1; 3% ±2; 1% ±1; 2% ±1; p 〉0.05), abnormal heads (mean 28%±13; 25%±16; 25%±17; 22%±16), distal droplets (mean 14%±10; 27%±12; 29%±15; 27%±16), proximal droplets (mean 15% ±6; 11% ±5; 11% ±6; 9% ±6), midpiece defects (mean 1% ±1; 2% ±2; 2% ±2; 3% ±2) and coiled tails (mean 1%±1; 1%±2; 1%±1; 1%±2). The only statistically significant difference was the percentage of sperm with bent tails between control group (mean 22%±17) and Sephadex^Ⓡ treatments (mean 8%±7; 7%±5; 8%±; p≤0.05) but not within Sephadex^Ⓡ treatments. For viability, a difference between control and Sephadex^Ⓡ treatments was observed (mean 52%±12; 66%±8; 57%±9; 57%±10; p≤0.05) and post hoc analysis showed the difference originating from Sephadex^Ⓡ G-25 (p≤0.05) with no difference between control and Sephadex^Ⓡ G-50 and G-75. Our results show that filtering frozen thawed epidydimal sperm through Sephadex^Ⓡ does not improve semen quality although when sperm was filtered through G-25 column there was a reduction in bent tails and an increase in viability. These results differ from other studies where fresh and cooled semen were used. Sephadex G-25 filtration can decrease the percentage of bent tails and increase percentage of viable sperm.