Abstract
The present study was conducted to evaluate the effect of accessory sperm cells that adhered to the zona pellucida or blastomeres on the accuracy of genetic diagnosis of preimplantation embryos. The properties of sperm cells as a template for DNA amplification were examined using bovine sperm cells frozen-thawed or incubated in PBS after thawing for 7 days at 39 C. Sexing by loop-mediated isothermal amplification (LAMP) and claudin-16 genotyping by polymerase chain reaction (PCR) were performed using 10, 50 and 100 sperm cells. When sexing based on LAMP was performed, no amplified DNA was detected in 10 sperm-derived samples, whereas male-specific (10-60%) and gender-natural DNA (30-100%) sequences were detected in 50 and 100 sperm-derived samples. The detection rates for gender-natural DNA sequences were higher in incubated sperm samples than in sperm samples immediately after freeze-thawing. The detection rates for claudin-16 were low (7-13%) regardless of the concentration of sperm cells and the period of incubation after thawing. The present results showed that male-specific DNA, gender-natural DNA and claudin-16 sequences were not usually amplified from a small number of sperm cells (≤10 cells). However, when a large number of sperm cells (≥50 cells) were present, male-specific and gender-natural DNA sequences were amplified at a high rate, and claudin-16 DNA sequences were also occasionally detected. These results raise the possibility that accessory sperm cells may reduce the accuracy of the genetic diagnosis of bovine embryos. Therefore, steps to prevent the contamination of sperm cells, such as removal of the zona pellucida and washing of sample blastomeres, are necessary to obtain an accurate result.
