Age- and -breed dependence of exogenous DNA binding to spermatozoa in goats

摘要

The purpose of this study was firstly to establish the technical protocols on optimization of goat Capra hircus spermatozoa binding exogenous DNA, and then to determine the ability of goat spermatozoa to bind foreign DNA from different breeds at 1–4 year-old. The ejaculates of three to four male goats, 2–3-year-old, were obtained by the artificial vagina method of collection with the aid of a female serving as dummy and then diluted with equal volume. The seminal plasma was removed by centrifugation. The linearized DNA plasmid was end-labeled. An orthogonal experimental L₁₆（4⁴）with four parameters in sperm-mediated gene transfer assays: DNA fragment sizes, the concentration of the foreign DNA, incubation time and temperature, was designed to optimize the method of goat sperm binding exogenous DNA. The positive goat spermatozoa were detected by in situ hybridization. A total of 149 goat sperm donors (21, 21, 19 and 10 Chuandong white goats of 1-, 2-, 3- and 4-year-old; 23, 22, 14 and 7 Boer×Nanjiang yellow F₁ goats, 6 Boer goats and 6 Nanjiang yellow goats, 2-year-old, respectively) was selected with the nearly sperm quality. The ejaculates of four breed male goats were obtained and screened by the method that established in this paper. On the condition of the seminal plasma sperm removed from freshly ejaculated semen by thorough washing, 1.00 μg linearized end-labeled DNA plasmid and 10⁶ spermatozoa incubated for 1 hour at room temperature, could have a good transfection rate. The efficiency of spermatozoa in picking up exogenous DNA of Chuandong white and Boer×Nanjiang yellow F₁ goats decreased with age. It was 39.34%±13.76% of Chuandong white goat at 1-year-old, and significantly decreased at 2- and 3-year-old (24.15%±12.46% and 23.40%±19.37%, P1 goats was 30.61%±21.59%, 20.75%±17.52%, 21.16%±13.98% and 22.88%±9.31% at 1-, 2-, 3- and 4-year-old, respectively. No significant difference was found among the ages ( P >0.05). Different goat breeds at 2-year-old exhibited different sensitivety to foreign genes. The sperm from Nanjiang yellow goat had the highest ability in picking up exogenous DNA, and that from Chuandong white goat also had a higher ability in picking up exogenous DNA. The positive sperm rates were 38.48%±17.54% and 34.62%±20.76% from these two breeds, respectively. No significant difference was found between the two breeds ( P >0.05). The sperm from Boer goat had the lowest ability in picking up exogenous DNA. The positive sperm rates were 13.85%±8.50%. The efficiency of goat spermatozoa in picking up exogenous DNA of is decreased with the age. Different goat breeds appeared to have different ability tp bind foreign genes [ Acta Zoologica Sinica 51（5）：1058–1066 ，2005].