Validation of flow cytometry for assessment of viability and acrosomal integrity of dog spermatozoa and for evaluation of different methods of cryopreservation.

The aims of the present study were: (i) to validate the accuracy of flow cytometry for assessment of viability and acrosomal status of canine spermatozoa; and (ii) to evaluate the cryopreservation protocols currently used for dog spermatozoa using flow cytometry. Data obtained by flow cytometry analysis of fresh dog spermatozoa stained with carboxyfluorescein diacetate (CFDA) and propidium iodide, or with fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and propidium iodide, were compared with those obtained by microscopic evaluation. The results demonstrated that flow cytometry is a precise method for evaluating the viability and acrosomal status of fresh samples of dog semen. A new triple staining procedure, using carboxy-SNARF-1, propidium iodide and FITC-PSA, was developed and was an efficient method for evaluating the following aspects of cryopreservation protocols for dog spermatozoa: (i) addition of 0.5% (v/v) Equex STM paste to a Tris-egg yolk-based extender; (ii) dilution of the semen in one or two steps; (iii) freezing semen by placing 0.5 ml straws horizontally above liquid nitrogen in a styrofoam box or lowering them vertically into a liquid nitrogen tank; (iv) thawing semen at two different rates; (v) packaging semen at different sperm concentrations; and (vi) diluting semen at different rates after thawing. The highest sperm survival and longevity was obtained when Equex was present in the semen extender, the semen dilution was performed in two steps to obtain a concentration of 2.0 x 10(8) spermatozoa ml-1, the freezing was carried out using the styrofoam box, the straws were thawed at 70 degrees C for 8 s and the semen was diluted 1:4 after thawing.