Vitrification of Large Volumes of Stallion Sperm in Comparison With Spheres and Conventional Freezing: Effect of Warming Procedures and Sperm Selection

Abstract

Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 μL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN2) and conventional freezing using 0.5 mL straws frozen in LN2 vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Significant higher values (P < .001) were obtained after vitrification using spheres in comparison with conventional freezing and vitrification in straws for TM (54.46 ± 3.2 vs. 36.47 ± 3.2 vs. 2.50 ± 1.2, %), PM (38.63 ± 3.4 vs. 15.11 ± 2.0 vs. 1.9 ± 0.9, %), IMS (65.40 ± 2.8 vs. 50.50 ± 2.8 vs. 21.63 ± 2.1, %), and AIS (48.89 ± 2.8 vs. 15.46 ± 1.7 vs. 4.69 ± 0.9, %). No differences were found between warming procedures. Single-layer centrifugation after warming at 42°C/20 seconds obtained higher values (P < .05) than unselected samples for TM (32.52 ± 5.8%), PM (14.22 ± 2.8%), IMS (60.01 ± 3.2%), and AIS (44.5 ± 2.2%), whereas selection after 60°C/15 seconds increased TM (23.11 ± 4.3%) and IMS (67.11 ± 3.9%). In conclusion, vitrification in spheres obtained better sperm quality than conventional freezing and vitrification in straws. Warming procedures did not affect the sperm quality but SLC could be a strategy to enhance the quality of the samples after sperm vitrification using 0.5 mL straws.