1. 124 Pretreatment of Dama gazelle (Nanger dama ruficollis) spermatozoa with cholesterol-loaded cyclodextrins improves cryosurvival.
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Wojtusik, Jessye, Songsasen, Nucharin, Penfold, Linda, Citino, Scott, and Pukazhenthi, Budhan S.
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PHYSIOLOGICAL effects of cholesterol , *SPERM motility , *CYCLODEXTRINS , *ACROSOMES , *CRYOPRESERVATION of organs, tissues, etc. , *ALIQUOTS (Chemistry) - Abstract
The Dama Gazelle, Nanger dama ruficollis, is critically endangered largely due to over-hunting and poaching. Fewer than 500 animals remain in the wild and ∼134 individuals are managed in captivity in North American zoos as a ’hedge’ against extinction. The ability to preserve and transport gene diversity could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to assess the beneficial effects of cholesterol-loaded cyclodextrin (CLC) on dama gazelle sperm cryosurvival. Electroejaculates from three male dama gazelle were evaluated for total motility, forward progressive status, concentration, and acrosomal membrane integrity. Ejaculates were diluted (120×106 sperm/ml) with Ham’s F10 supplemented with Hepes (Irvine Scientific), aliquoted into five 1.5ml eppendorf centrifuge tubes and incubated (37°C; 15min) with 0, 0.5, 1.5, 3.0 and 6.0mg/ml CLC, respectively. Aliquots were centrifuged (300g; 8min) and the resulting sperm pellets were resuspended (250×106sperm/ml) in TEST-Yolk Buffer Refrigeration Medium (Irvine Scientific) containing 6% (v/v) glycerol. Sperm suspensions were loaded in straws (0.25ml), cooled to 5°C and placed 4cm over liquid nitrogen (LN2) for 10min before being plunged into LN2. Straws were thawed at 37°C (30s), stored protected from light at 25°C and evaluated for total motility, progressive status, and acrosomal integrity at 0 (immediately post-thaw), 1, 2 and 3h. Data were analyzed using generalized linear regression models and significance was defined as P <0.05. All males produced ejaculates with good initial motility (∼80%), progressive status (∼3.3; scale 0–5; 5=best), intact acrosomes (∼92.5%) and morphologically normal spermatozoa (∼65%). Immediately after thawing, sperm motility was unaffected (P <0.05) in the presence of 0.5mg/ml CLC (73.3±2.6%) but declined (P <0.05) in other groups (0, 56.7±3.1; 1.5, 60±0.8; 3.0, 41.7±6.9; and 6.0, 40±0.8%). At 1 h post-thaw, there were no differences in sperm motility amongst 0, 0.5, 1.5 and 3.0mg/ml CLC but 6.0mg/ml CLC was lower (P <0.05) than 0, 0.5, and 1.5mg CLC/ml. At 2h post-thaw, aliquots treated with 0.5 CLC exhibited the highest (P <0.05) motility. At time 3h post-thaw, sperm motility was higher in 0.5mg/ml CLC than non-treated control and 6mg/ml CLC but comparable to 1.5 and 3.0mg/ml CLC treated spermatozoa. There were no differences amongst 0, 1.5, 3.0 or 6.0mg/ml CLC. Progressive status was unaffected by treatment at all times evaluated, but declined (P <0.05) over time (0h, 3.8±0.2; 3h, 2.8±0.3) within all treatments. Acrosome integrity did not differ from raw ejaculates amongst treatment or time. Data demonstrate that pre-treatment of dama gazelle spermatozoa with CLC (0.5mg/ml) prior to cryopreservation protects spermatozoa from cryo-injury. These data could facilitate the development of artificial insemination technologies and establishment of a frozen repository for this species to assist with establishing self-sustaining populations and supporting reintroduction projects. Source of funding: None declared. Conflict of interest: None declared. pukazhenthib@si.edu [ABSTRACT FROM AUTHOR]
- Published
- 2013
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