303 EFFECT OF THE PRESENCE OF EXOGENOUS DNA AND RECOMBINASE-A PROTEIN ON THE BOAR SPERM FUNCTIONALITY

Abstract

Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer based on the intrinsic ability of sperm cells to bind exogenous DNA molecules and to transfer them into the oocyte. It has been reported that coating exogenous DNA with recombinase-A protein (RecA) may improve the efficiency of transgenic embryo production (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338). The objective of this study was to investigate the effect of the presence of DNA and DNA-RecA complex on the sperm functionality. Spermatozoa were incubated with linealized plasmid 5.7 kb enhanced green fluorescent protein (DNA) or RecA-DNA complexes (40:1 or 40:3 protein:DNA w/w) in a relation of 10⁸ cells mL^–1 and 5 to 15 μg of DNA mL^–1. Sperm viability, membrane lipid disorder, reactive oxygen species (ROS) generation, and motion parameters were evaluated by flow cytometry and computer-assisted semen analysis (CASA) for control (no DNA), DNA (5 μg), RecA 5 (5 μg), and RecA 15 (15 μg). A total of 5 replicates were used. Data were analyzed by ANOVA. The sperm viability measured by propidium iodide staining was significantly reduced by the presence of RecA DNA complexes (viable cells 77.5^a v. 69.4^ab v. 55.6^b v. 35.8^c%; P < 0.01). Lipid membrane disorder (measured by merocyanine 540 and Yopro 1 staining) were significantly reduced in the group with high exogenous DNA concentration (RecA 15 group), with a reduction of percentage of viable cells and low lipid disorder (67.4^a v. 60.0^a v. 51.5^a v. 24.3^b; P < 0.01). However, the ROS generation (measured by 2′,5′-dichlorofluorescein diacetate) was not different in the different experimental groups (fluorescent units 20.3 v. 25.5 v. 26.2 v. 13.9; P = 0.36). The motion parameters measured by CASA were affected by the presence of DNA-RecA complexes with a significant reduction in percentage of motile and progressive motile cells in the group RecA 15 (% progressive motility 46.7^a v. 43.9^a v. 46.8^a v. 20.3^b; P < 0.01). Also, it was detected a change in the pattern of motility with an increase in the sperm velocities (velocity average path, velocity straight line, and VCL) in groups DNA and RecA 5 and a reduction of these values when the concentration of DNA was 15 μg mL^–1 (VCL μm s^–1 97.5^a v. 106.4^b v. 109.1^b v. 58.1^c; P < 0.01). The motions parameters (LIN, STR, WOB, ALH, BCF) showed significant lower values for the group RecA 15. These results shown that the presence of DNA and RecA-DNA complex modified the pattern of motility and reduced the viability and functionality of the boar sperm in a concentration dose way. It is necessary to select an adequate DNA and RecA concentration that would protect the sperm functionality and optimize the binding process.

Supported by BIOCARM 10BIO2005/01-6463 and AGL2006-03495.