Japanese Journal of Veterinary Research;Volume 64 Number 4


Cryopreservation of stallion semen: laboratory assessment of sperm injuries after cushioned centrifugation and freezing with conventional and alternative directional freezing methods

Puglisi, Roberto;Bornaghi, Valeria;Severgnini, Alex;Vanni, Roberta;Balduzzi, Donatella;Galli, Andrea

Permalink : http://hdl.handle.net/2115/63763
JaLCDOI : 10.14943/jjvr.64.4.235
KEYWORDS : cushioned centrifugation;flow cytometer;mitochondrial status;membrane status;kinetics


Fresh 36 ejaculates of 13 stallions were split into two volumes, centrifuged with and without cushion and frozen with Conventional and two prototype, Drum and Directional, methods using 0.5 ml straws for the Conventional and Drum, and 2 ml flat straws for both the Drum and Directional. Cushioned centrifugation increased total motility (61.2 ± 18.6% vs. 57.5 ± 18.6%; P < 0.001) and mean velocity (84.3 ± 15.6% vs. 83.2 ± 13.8%; P < 0.05) when compared to not cushioned centrifugation, estimated after cooling the sperm at 4℃ for 90 min before freezing. Cushioned centrifugation also increased (P < 0.001) spermatozoa with polarized mitochondrial membranes (46.8 ± 11.4% vs. 43.4 ± 10.6%) and intact plasmatic/acrosomal membranes (41.0 ± 11.2% vs. 38.5 ± 11.3%) of frozen/thawed sperm, with respect to not cushioned centrifugation. However, no effects of the centrifugation were evidenced for classical kinetic parameters. Flat straws had negative effect for almost all the parameters analyzed at thawing (T0) and after 3 hours' incubation at 37℃ (T1), while the Drum method with Paillettes did not show appreciable affects. The variability among stallions was relevant (5% to 69% variance for kinetics and membrane status), while the variability among ejaculates was minor (9% to 28%). Factorial analysis identified three relevant factors with different informational content: Factor 1 represented by membranes status, Factor 2 by kinetics estimated at T0, and Factor 3 by kinetics estimated at T1. Cushioned centrifugation had some beneficial effects for the membrane status of the frozen/thawed sperm, while the use of flat straws needs to be improved.