Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa

Abstract

Prostatic fluid is unsuitable for preserving dog semen at 4 °C and exerts harmful effects upon the spermatozoa during the freezing process. Centrifugation immediately after sperm collection is a common method to remove prostatic admixture. In the present study, dog semen, diluted to 25×10 6/ml, was exposed for 5 min to four different centrifugation speeds (180× g, 720× g, 1620× g and 2880× g) to determine subsequent sperm losses in the supernatant and to assess sperm survival over time. Using 180× g as centrifugation speed, 8.9% of the sperm cells was lost upon supernatant removal. Using 720× g, 1620× g or 2880× g, sperm losses were lower, 2.3, 0.4 and 0.006%, respectively. After centrifugation, the sperm pellet was rediluted in egg–yolk–Tris extender, cooled and stored for 3 days at 4 °C. Motility, progressive motility, membrane integrity and sperm morphology were assessed daily. Acrosomal status was assessed after 3 days of storage. The only functional parameter which was influenced by centrifugation speed was membrane integrity as evaluated by means of SYBR14-PI staining: significantly more dead and moribund sperm cells were found after centrifugation at 1620× g and 2880× g after 48 and 72 h of storage at 4 °C. When higher initial sperm concentrations (50×10 6, 75×10 6 or 100×10 6/ml) were evaluated for sperm losses, less than 2.3% of the initial total sperm cells was lost at lower centrifugation speeds. We conclude that centrifuging dog sperm for 5 min at 720× g is the best strategy to remove prostatic fluid because the loss of sperm cells is acceptable and the functional parameters of the spermatozoa are well preserved, even after 3 days of storage.