2 research outputs found
USE OF COMBINATION OF FLUORESCENT PROBES TO IDENTIFY SPERM SUBPOPULATIONS FOR QUALITY ASSESSMENT OF FRESH AND CRYOPRESERVED CANINE SEMEN. PRELIMINARY RESULTS
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.聽El uso de marcadores fluorescentes en la evaluaci贸n de la morfofisiolog铆a esperm谩tica permite una mayor precisi贸n, comparada con la naturaleza subjetiva de algunas pruebas de rutina en la valoraci贸n del semen. En este estudio se us贸 la combinaci贸n de pruebas fluorescentes: yoduro de propidio; Hoechst 33342 y FITC-PSA en semen fresco y descongelado de perro, para la identificaci贸n de las siguientes subpoblaciones morfol贸gicas: II (membranas plasm谩tica y acrosomal intactas), IL (membrana plasm谩tica intacta y membrana acrosomal da帽ada), LI (membrana plasm谩tica da帽ada y membrana acrosomal intacta) y LL (ambas membranas da帽adas). Cuando se comparan los resultados obtenidos con los resultados de las pruebas usadas convencionalmente en la evaluaci贸n seminal (motilidad y vigor esperm谩ticos, prueba hipoosm贸tica y alteraciones morfol贸gicas), se observ贸 poca correlaci贸n. La poblaci贸n II disminuy贸 desde el semen fresco al descongelado, mientras que la poblaci贸n LL se increment贸 (p<0.05). la poblaci贸n IL estuvo compuesta por un n煤mero extremadamente peque帽o de c茅lulas, pero incremento (p<0.05) desde el semen fresco al descongelado. En el semen descongelado los defectos mayores tuvieron una correlaci贸n positiva con la poblaci贸n LL (p<0.01). En el semen descongelado, los resultados de la prueba hipoosm贸tica (n煤mero de c茅lulas que reaccionan al medio) se correlacionaron positivamente con la poblaci贸n II (p<0.05). El uso de esta asociaci贸n de pruebas fluorescentes permiti贸 la valoraci贸n y la cuantificaci贸n m谩s precisa de los da帽os a la membrana plasm谩tica y acrosomal mediados por la criopreservaci贸n.
Ocelot and oncilla spermatozoa can bind hen egg perivitelline membranes
We evaluated the capacity of ocelot and oncilla spermatozoa to bind to the perivitelline membranes (PVMs) of hen eggs in a sperm binding assay (S-PVM). In addition, a device that improves the standardization of the assay was developed. The number of sperm bound to the PVM in fresh (T1) and frozen鈥搕hawed (T2) semen from both species was compared to the sperm quality observed in routine tests. The PVM was stretched on a circular silicone device to create a standardized area for analysis. In both treatments and for both species, the spermatozoa were able to bind to the PVM, indicating that PVM may be used for a sperm binding assay in ocelot and oncilla. The S-PVM assay did not differ in fresh and frozen鈥搕hawed ocelot sperm (p > 0.05). However, fewer oncilla sperm (p < 0.05) were bound to the PVM in T2, indicating that the proposed test may be able to detect injuries that compromise sperm binding abilities. The device maintained the PVM stretched during the processing and defined the evaluation area