Prediction of Fetal Sex by Amplification of Fetal DNA Present in Cow Plasma

Abstract

Abstract. The aim of the present study was to predict fetal sex at different time points of gestation in cattle by detecting the fetal SRY gene in cow plasma. Plasma DNA was extracted from the blood samples of 110 pregnant cows during the gestational period of 30 to 242 days. Nested PCR was employed to detect the fetal SRY, which the male fetus carries exclusively, in cow plasma. The cows positive for SRY were predicted to carry male fetuses. The results showed that the fetal DNA from cow plasma was successfully amplified and that fetuses could be sexed with an overall accuracy rate of 100% (43/43) for males and 91.0% (61/67) for females and with accuracy rates of 100% (3/3) for males and 85.7% (12/14) for females at 30 EN 59 days of gestation and 100% (40/40) for males and 92.5% (49/53) for females at more than 2 months of gestation, respectively. This suggests that the molecular method developed here could be used in sex prediction for fetuses. Key words: Cow plasma, Fetal Sex, Nested PCR, Sry (J. Reprod. Dev. 56: [639][640][641][642] 2010) he discovery of fetal DNA (fDNA) in maternal circulation [1] and transcervical cells has led to noninvasive prenatal genetic diagnosis and sex prediction for fetuses during early pregnancy in women [2]. Although it was recently demonstrated that fDNA in women and rhesus monkeys could be used as a tool for fetal sex determination as early as at the 5th gestational week using the Y chromosome DNA sequence as a probe for screening male fetuses with an accuracy rate of 100% [3][4][5][6], little is known about fDNA in domestic animals. In 1996, Kadokawa et al. believed, based on their previous research, that fetal cells were very rare or may be absent in bovine maternal blood and that they could not be used for prenatal sexing by a PCR method [7]. However, Xi et al. reported in 2006 that they had successfully predicted fetal sex by amplifying the SRY gene from pregnant cow blood [8]. However, only 30 cows were tested in their report, and the overall accuracy rate was 80%; the accuracy rate for 30-59 days of gestation was 60% (6/10), and they also did not analyze the sequences of the PCR products.The aim of present study was to verify the possibility of detecting fDNA in cow plasma and develop a noninvasive method of fetal sex identification in pregnant cows in order to offer a way for sex selection in dairy production and for early selection of cattle in breeding programs as well as for elimination of harmful genes in bulls in the fetal phase. Materials and Methods Blood samples and DNA isolationTen ml of blood was collected from each of 110 pregnant Holstein cows that were 3-6 years of age and at 30-242 days of gestation through the vena caudalis by single-use syringe. All the blood samples were put into acid citrate dextrose anticoagulant tubes and centrifuged at 4 C and 1600 × g for 10 min. Supernatants were stored at -20 C for further analysis. Blood samples from 2 virgin heifers at 3 months of age with no history of contact with bulls or semen and 6 bulls that were 5 yea...

Cell-free DNA release by mouse placental explants

“…In pregnant mice, Khosrotehrani et al [29] described an increase in cell-free fetal DNA levels during each of the 3 weeks of gestation. Wang et al [30] reported the presence of cell-free fetal DNA in the plasma from pregnant cows, and de Leon et al [31] made similar observations in pregnant horses. In pregnant sheep, Kadivar et al [32] detected a 1.65-fold increase in maternal plasma cell-free fetal DNA levels for the last two months of gestation compared to the middle months.…”

Testis-Specific Protein Y-Encoded Copy Number Is Correlated to Its Expression and the Field Fertility of Canadian Holstein Bulls

“…Intron-containing pseudogenes have also been reported for TSPY (known as TSPY-C ) [Verkaar et al, 2004] so to avoid amplification of these pseudogenes, the genomic primers used in this study were designed in regions of low sequence similarity between TSPY-C and TSPY . The sex-determining region, SRY , was selected as a single copy Ychromosomal reference gene [Chandler et al, 2007], using primer sets SRY-1 [Hamilton et al, 2009] and SRY-2 [Wang et al, 2010] for trials #1 and #2, respectively.…”

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

“…Currently, there are at least four approaches to sex determination in ruminant species, based on the onset of development: a) artificial insemination using flow cytometry sexsorted semen (De Vries et al, 2008); b) polymerase chain reaction (PCR)-based detection of gender-specific sequences in preimplantation embryos prior to embryo transfer Tsai et al, 2011); c) ultrasound fetal scanning of pregnant females in early pregnancy (Hughes and Davies, 1989;Davis and Haibel, 1993); and d) fetal DNA detection in the blood/serum of pregnant females (Wang et al, 2010;Kadivar et al, 2013). Each approach has its technical and economic advantages but also its limitations, and the choice of a particular procedure may depend on many factors.…”