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International Journal of Morphology

On-line version ISSN 0717-9502

Abstract

SALINAS, Paulo; PEZO, Felipe; SANCHEZ, Raúl  and  RISOPATRON, Jennie. Validation of SYBR-14 and 6-CFDA to Evaluate the Viability and Plasma Membrane Integrity in Sperm of Chihuahua Canine Breed. Int. J. Morphol. [online]. 2014, vol.32, n.1, pp.16-21. ISSN 0717-9502.  http://dx.doi.org/10.4067/S0717-95022014000100003.

In applying reproductive techniques in vitro it is important to determine the fertilizing capacity of the sperm, for this a combination of dyes were used to assess different parameters of sperm function, thereby increasing the accuracy of the estimation of the sample. In dogs (Canis lupus familiaris) Chihuahua breed there is no precedent for evaluating sperm function parameters. The aim was to assess the viability and plasmatic membrane integrity, basic parameters of sperm function. Propidium iodide (PI) was used, a fluorescent dye-specific DNA, which combined with fluorochromes permeable acts as marker of the sperm membrane integrity. The effectiveness of the use of 6-CFDA and SYBR-14 fluorochromes combined with PI was also compared to determine viability and sperm membrane integrity using flow cytometry. Fresh semen of dogs (n=5) Chihuahua breed was used with a concentration of >200x106 sp/ml and progressive motility >80%. Three protocols were performed: group 1: SYBR-14/PI, group 2: 6-CFDA/PI and group 3: PI. The plasma membrane integrity of sperm was similar, independent of the fluorophore used between groups 1 and 2 (13.9±37.26 and 33.8±14.6, respectively, p=0.4601). This also applied to sperm viability between groups 1, 2 and 3 (62.7±13.9, 66.1±14.6 and 66.4±13.3, respectively, p=0.8987). No difference was demonstrated in effectiveness to determine the viability and integrity of the sperm membrane using SYBR-14 and 6-CFDA, both dyes can be incorporated in to routine analysis of semen in canine Chihuahua breed.

Keywords : Canine; Chihuahua; Semen; Sperm; SYBR-14; 6-CFDA; Flow cytometry.

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