Influence of two extenders used for short-cooled storage on motility of Quarter Horse semen submitted to a test freezing

Abstract

Identifying the most appropriate extender for short-cooled storage is essential for maximizing post-thawing results. The objectives of this study were assessing the impact of two extenders routinely used for cooled-storage on post-thaw progressive motility (PM) and identifying the most adequate freezing extender for these samples’ cryopreservation. One ejaculate from each of ten Quarter Horse stallions, routinely used in a reproduction center, was collected. Samples were randomly diluted in Botu-Semen (BS) (G1) (n=5) or EquiPlus (EP) (G2) (n=5) to 50 × 106 cells/ml and maintained at 5°C for 8 h, before being equally split into 9 tubes. They were centrifuged (600g x 20 min) and the pellet was resuspended with one of 9 different freezing extenders (Blue, Green, Orange, Red, Violet, Star, MX3, Gent and Botucrio) to 200 × 106 sperm/ml. Then straws were manually filled and kept at 5°C for 20 min in a refrigerator. Subsequently, they were exposed to nitrogen vapors for 20 min, before immersion in liquid nitrogen. Straws were thawed in a water bath at 37°C for 30 seconds. Evaluation of sperm PM after cooling storage (t0) and after thawing (t1) was performed using the CASA System (AndroVision, Minitube, Germany). A generalized linear model was used, being the response variable, PM, and the fixed effects, cooling extender (two levels: G1 and G2) and freezing extender (9 levels: each freezing extender). To analyze the number of samples with post-thaw PM ≥ 30% according to the cooling extender, a generalized linear model was used too. Differences were considered significant when P values were <0.05. Values are expressed as mean ± SD (Graph Pad, USA). Mean PM before freezing did not show significant differences between G1 and G2 (73.4±14.4% vs 72.8±4.3%, respectively). Post-thaw PM was significantly greater in samples of G2 vs. G1 (28.6±10.2% vs 16.15±10.2%) (P˂0.001). Moreover, 48.8% of samples of G2 showed post-thaw PM˃30%, while only 8.8% of G1 did (P˂0.01). Considering each stallion's performance, 5/5 individuals of G2 reached a post-thaw PM˃30%, while only 2/5 of G1 did. Samples of G2 showed a greater post-thaw PM when using STAR, Orange and Violet, compared to G1 (34.6±6.3% vs. 18.7±11.9%, 23.8±8.2% vs. 11.8±5.8% and 38.3±6.7% vs. 19.1±8.9%, respectively). Post-thaw PM˃30% was attained for 4/5 stallions of G2 whose sample was diluted in STAR and Botucrio, and for 5/5 whose sample was diluted in Violet. Only 1/5 stallions of G1 attained post-thaw PM˃ 30% (using STAR, Botucrio, Green and Violet). No interaction was observed between the cooling extender and the freezing extender. In general, mean PM decreased from t0 (73.1±10.1%) to t1 (22.4±11.9%) (P˂0.001). In conclusion, PM after-thawing was better when samples were stored in EP than in BS. Better results were accomplished when using Violet, Star and Botucrio as freezing extenders.