การประยุกต์ใช้เทคนิคปฏิกิริยาลูกโซ่โพลิเมอเรสแบบมัลติเพล็กซ์ในเวลาจริงสำหรับประเมินสัดส่วนเพศอสุจิในน้ำเชื้อพ่อม้า

Abstract

The purposes of this study were to apply a multiplex real-time polymerase chain reaction technique for estimating the sperm sex ratio of stallion semen and to test the performance and validate the technique with known DNA sample ratios. Firstly, fresh semen samples from five stallions were examined for semen quality by using a computer-assisted sperm analysis (CASA). The results revealed that the number of sperm cells, movement rate, and survival rate of sperm cells showed high quality, which could be tested for further experimentation. The present study was divided into two experiments. In experiment 1, a primer set that was specific to the PLP gene on the X chromosome and the SRY gene on the Y chromosome was designed, and the efficacy of each primer set was examined by using the PCR technique. Moreover, the performance of the specific primer and probe of the SRY gene and PLP gene was then confirmed by using singleplex real-time PCR technique and multiplex real-time PCR technique, respectively. In the real-time PCR method, the TaqMan probe specific to the SRY gene was labeled with the FAM fluorescent dye, and the PLP probe was labeled with the HEX fluorescent dye. The fluorescent signal of SRY gene (FAM) and PLP gene (HEX) were shown when tested with male peripheral white blood cells (XY). While female white blood cells (XX) illustrated only HEX signal. The SRY2 and PLP3 specific primers that were used in this study showed high efficiency when both of them were gathered into a single reaction. To construct the standard curve, the six concentrations of white blood cell DNA that were prepared in 5-fold dilutions from male horse lymphocytes were as follows: 1,000 ng/uL, 200 ng/uL, 40 ng/uL, 8 ng/uL, 1.6 ng/uL and 0.32 ng/uL. The straight-line slope values of SRY gene and PLP gene were calculated in the range of -3.47 and -3.56, respectively, representing the accuracy of quantification of the new amplicons at 93%. Meanwhile, the standard curve from plasmid DNA showed the straight-line slope of the SRY gene and PLP gene was in the range of -3.51 and -3.60, respectively, representing an accuracy of 91.5%. In experiment 2, the developed standard curves (white blood cell DNA and plasmid DNA) were used to calculate the sperm sex ratio with the following samples: 1) plasmid DNA with a known ratio, 2) DNA from horse lymphocytes with a known ratio, and 3) normal semen. It was found that the Y-spermatozoa in stallion semen (%Y) were accurate at 95% CI and the evaluation of sex ratio X : Y was tested by using the two-tailed paired t-test. The data revealed that the sperm sex ratio was not statistically significant (P > 0.05). Thus, this study concluded that the application of multiplex real-time polymerase chain reaction technique with male horse white blood cells DNA could be used to create a standard curve that reduced the cost and time-consuming when compared with plasmid DNA. Also, this application could be used as a standard method for determining the sperm sex ratio of stallion semen (quality control method) and verifying the accuracy of semen in the laboratory prior to artificial insemination in field trials.