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Use of Combination of Fluorescent Probes to Identify Sperm Subpopulations for Quality Assessment of Fresh and Cryopreserved Canine Semen. Preliminary Results
A.C. Csermak Jr., T.A. R. de Paula*...
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Abstract
The use of fluorescent markers in the evaluation of sperm morphophysiology allows a better accuracy, compared to the subjective nature of some routine tests in semen qualification. In this study was used the combination of fluorescence probes: propionate iodide, Hoechst 33342 and FITC-PSA in fresh and thawed dog semen, to the identification of the following morphological subpopulations: II (intact plasma and acrosomal membranes), IL (intact plasma membrane and lesioned acrosomal membrane), LI (lesioned plasma membrane and intact acrosomal membrane) and LL (both membranes lesioned). When comparing the results obtained with the results of the tests used conventionally in semen evaluation (sperm motility and vigor, hypoosmotic test and morphological alterations), little correlation was observed. The II population declined from fresh semen to thawed, while LL population increased (p <0.05). The IL population was composed of extremely small numbers of cells but increased (p <0.05) from fresh semen to thawed semen. In the thawed semen the major defects had a positive correlation with the LL population (p <0.01). For the thawed semen, the results of the hypoosmotic test (number of cells that reacted to the medium) correlated positively with population II (p <0.025), that is, different from that observed in fresh semen. Although all tests were able to detect decrease in sperm quality post-thawing (p <0.05). The use of this fluorescent probe association allowed qualification and more accurately quantification of plasma membrane and acrosomal insults mediated by cryopreservation.
Resumen
El uso de marcadores fluorescentes en la evaluación de la morfofisiología espermática permite una mayor precisión, comparada con la naturaleza subjetiva de algunas pruebas de rutina en la valoración del semen. En este estudio se usó la combinación de pruebas fluorescentes: yoduro de propidio; Hoechst 33342 y FITC-PSA en semen fresco y descongelado de perro, para la identificación de las siguientes subpoblaciones morfológicas: II (membranas plasmática y acrosomal intactas), IL (membrana plasmática intacta y membrana acrosomal dañada), LI (membrana plasmática dañada y membrana acrosomal intacta) y LL (ambas membranas dañadas). Cuando se comparan los resultados obtenidos con los resultados de las pruebas usadas convencionalmente en la evaluación seminal (motilidad y vigor espermáticos, prueba hipoosmótica y alteraciones morfológicas), se observó poca correlación. La población II disminuyó desde el semen fresco al descongelado, mientras que la población LL se incrementó (p<0.05). la población IL estuvo compuesta por un número extremadamente pequeño de células, pero incremento (p<0.05) desde el semen fresco al descongelado. En el semen descongelado los defectos mayores tuvieron una correlación positiva con la población LL (p<0.01). En el semen descongelado, los resultados de la prueba hipoosmótica (número de células que reaccionan al medio) se correlacionaron positivamente con la población II (p<0.05). El uso de esta asociación de pruebas fluorescentes permitió la valoración y la cuantificación más precisa de los daños a la membrana plasmática y acrosomal mediados por la criopreservación.
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Affiliation of the authors at the time of publication
Departamento de Veterinária, Viçosa, Minas Gerais Brasil 36570-000. Correspondence should be addressed to (La correspondencia debe dirigirse a): T.A.R de Paula, e-mail: [email protected]
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