The effect of methyl-beta-cyclodextrin on DNA absorption and quality of posttransfected sperm

Abstract

Sperm can be selected as a natural vector for the production of transgenic animals. Methyl-beta-cyclodextrin (MBCD) removes cholesterol from the phospholipid membrane of sperm and improves the efficiency of DNA uptake by sperm. In experiment 1, fresh sperm was treated with various concentrations of MBCD. The direct effects of MBCD on sperm parameters were monitored. In experiment 2, different concentrations of MBCD (0, 1, 2, and 4 mmol) were assessed for the transfection of genetically exogenous construction to rooster sperm. Washed semen was divided into 5 equal groups for the incubation and transfection with a pcDNA3.1+/hG-CSF vector (exogenous DNA) as follows; Treatment I—Control (washed semen without DNA); Treatment II—Control (washed semen with DNA); Treatment III—(washed semen incubated with DNA and 1 mmol MBCD); Treatment IV—(washed semen incubated with DNA and 2 mmol MBCD); and Treatment V—(washed semen incubated with DNA and 4 mmol MBCD). We demonstrated that rooster spermatozoa spontaneously can uptake exogenous DNA; this was assessed using exogenous DNA amplification (sperm genomic DNA used as a template for PCR reaction) after DNase I treatment. In addition, total motility (TM), progressive motility (PM), velocity parameters [curvilinear velocity (VCL), straight linear velocity (VSL), sperm track straightness (STR), linearity (LIN)], membrane integrity (MI), and membrane functionality were posttransfectionally evaluated. The concentrations of 1 and 2 mmol MBCD significantly (P < 0.05) improved the motion characteristics and membrane integrity of fresh sperm. The presence of hG-CSF in rooster sperm was detected by PCR and based on sperm analyses MBCD (1 mmol) improved the percentage of motility (98.9 ± 0.81), membrane functionality (64 ± 1.64), and MI (76.2 ± 1.65) after transfection when compared with the other groups (P < 0.05). For the production of transgenic chicken, hens were inseminated (AI) by transfected sperm treated with 1 and 0 mmol MBCD. A PCR analysis of the blood samples and dead embryo tissues of chicks did not reveal the transgene integration.