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Doctoral thesis, 2000

Flow cytometry in the assessment of fresh and frozen-thawed dog semen, and the effects of different cryopreservation methods on post-thaw sperm survival and longevity

Peña Martínez, Ana Isabel


The use o f flow cytometric methods for assessing sperm viability and acrosomal status of dog spermatozoa might facilitate the improvement and development of new cryopreservation protocols and allow accurate comparisons between different freezing treatments, since flow cytometry provides means o f sperm assessment that are more accurate than conventional assays. The main objectives o f the present studies were a) to validate the accuracy o f flow cytometry techniques to assess viability and acrosomal status o f canine spermatozoa separately or simultaneously and b) to evaluate cryopreservation protocols currently used for dog spermatozoa using flow cytometry.

The plasma membrane integrity of fresh dog spermatozoa stained by carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) was evaluated in the first study. Data obtained by flow cytometry analysis were compared with those obtained by microscopic evaluation under epifluorescence illumination and by phase contast evaluation o f the samples stained with eosin-nigrosin. High correlation coefficients were found between the flow cytometry procedure and the 2 microscopy techniques. The results of this study validated the use o f flow cytometry as a precise method for assessing the viability o f dog spermatozoa. A procedure to simultaneously assess viability and acrosomal integrity of freshly ejaculated dog sperm by flow cytometry was developed in the second study. Sperm cells were stained with fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and PI. Dog ejaculates were divided in aliquots and treated with different concentrations o f lysophosphatidylcholine (LPC) to artificially induce the acrosome reaction in different proportions of spermatozoa. Data obtained by flow cytometric analysis o f each sample were compared with those obtained by microscopic evaluation under epifluorescence illumination and by light microscopy evaluation of smears stained with SpermacR staining. The results proved flow cytometry to be a precise method for evaluating the viability and acrosomal status of fresh dog semen samples. A new triple staining procedure was developed in the third study to assess frozen-thawed dog spermatozoa, where, in addition two cryopreservation methods (Andersen and CLONE) were compared. Plasma membrane integrity and acrosomal status of spermatozoa were evaluated simultaneously by flow cytometry using the new combination o f 3 fluorescent dyes: Carboxy-SNARF-1, to identify the live spermatozoa, PI to identify the dead or dying cells and FITC-PSA to detect acrosomal damage to spermatozoa. This new triple staining method provided an efficient procedure for evaluating frozen-thawed dog semen samples when using flow cytometry or fluorescence microscopy. Motility and plasma membrane integrity of spermatozoa immediately postthaw were similar for the two cryopreservation methods, but the proportion o f damaged acrosomes after thawing was lower, and the spermatozoa had a higher thermoresistance, when using the Andersen method than when using the CLONE method.

In the two last studies, different aspects of the cryopreservation protocol for dog spermatozoa were investigated, using flow cytometry and the triple staining procedure previously described to evaluate differences between the different treatments. In the fourth study, we evaluated the effects and interactions of: 1) adding 0.5% Equex STM paste to a Tris-egg yolk-based extender, 2) diluting the semen in 1 step (adding the Trisegg yolk-based extender containing 5% glycerol and 0.5% Equex before the equilibration period) or in 2 steps (adding a first Tris-egg yolk-based extender containing 3% glycerol and 0% Equex before the equilibration period, and a second Tris-egg yolk-based extender containing 7% glycerol and 1% Equex after the equilibration period), 3) freezing according to 2 methods (placing the 0.5-mL straws horizontally 4 cm above liquid nitrogen in a styrofoam box or lowering them vertically into a LN2 tank in 3 steps) and 4) thawing at 2 rates (70 °C for 8 sec and 37 °C for 15 s ). The best post-thaw survival and thermoresistance o f spermatozoa was obtained when Equex was present in the extender, the semen dilution was performed in 2 steps, the freezing was carried out using the styrofoam box and the straws were thawed at 70 °C for 8 s instead o f at 37 °C for 15 s. In the fifth study, the effects o f freezing doe semen with different sperm concentrations (50 x 10^, 100 x 10^, 200 x 10^ and 400 x 10® spermatozoa/ mL) and diluting the semen postthawing at different rates (1:0, 1:1, 1:2 and 1:4) were assessed. The best longevity was obtained when semen packaged at a concentration of 200 x 10^ spermatozoa/ mL was diluted after thawing at 1: 4 dilution rate.


dog spermatozoa; fluorescent staining; flow cytometry; cryopreservation

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria
2000, number: 71
ISBN: 91-576-5912-5
Publisher: Swedish University of Agricultural Sciences

Authors' information

Peña Martínez, Ana Isabel
Swedish University of Agricultural Sciences, Department of Obstetrics and Gynaecology

UKÄ Subject classification

Clinical Science

URI (permanent link to this page)