Evaluation of deoxyribonuclease activity in seminal plasma of ejaculated chicken semen.
OBJECTIVE
To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents.


METHODS
A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 degree C for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light.


RESULTS
The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L approximately 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents.


CONCLUSIONS
The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.
Sato
Fuminori
F
Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka 8128581, Japan.
Soh
Tomoki
T
Hattori
Masa-Aki
MA
Fujihara
Noboru
N
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002645:Chickens / Q000502:physiology*; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000032:analysis*; D004492:Edetic Acid / Q000494:pharmacology; D006358:Hot Temperature; D007202:Indicators and Reagents; D008297:Male; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000201:enzymology*
D002413:Cations, Divalent; D017931:DNA Primers; D007202:Indicators and Reagents; D004247:DNA; D004492:Edetic Acid; D003851:Deoxyribonucleases