First report of DNA detection and sex determination in leaked fluid after needle collapse of large equine blastocysts

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The current method for sex determination of equine blastocysts involves aspiration of blastocoel fluid followed by PCR, which can be challenging due to the need for specialized equipment and often requires the embryo to be shipped to a separate lab for analysis. In this study, we investigated a new method for sex determination using leakage from manual collapse prior to vitrification, a technique which previously resulted in a pregnancy in our laboratory (1 of 3, unpublished results). We hypothesized that leaked blastocele fluid into the surrounding medium could provide sufficient DNA for amplification and sex determination. From a total of 14 embryos (900 to 1200 μm diameter), 4 were collapsed by micromanipulator-assisted aspiration (Micro group), and 10 were collapsed by manual puncture (Needle group) using a 32-G, penta-point needle (BD Nano, Canada). Additional blastocoel leakage (up to 80% of original volume) was obtained by forced pipetting using a 275 μm flexipet. All manipulations were conducted in TALP without serum. Samples were snap-frozen (30 μl) and stored at -80°C. DNA amplification was performed by multiplex PCR targeted to sex chromosome-linked zinc finger protein genes (ZFx/ZFy) for both sex DNA detection and the Y-encoded testis-specific protein (TSPY) for Y chromosome detection. The primer sequences were ZFx/y-F 5’-ATAATCACATGGAGAGCCACAAGCT-3’, ZFx/y-R 5’-GCACTTCTTTGGTATCTGAGAAAGT–3’, and TSPY-F 5′-GAAGTCAGGCACACCAGTGA-3′, TSPY-R 5′-TAAGGCTGCAGTTGTCATGC-3′, with 445bp and 280bp products, respectively. The reaction was carried out in a 25 μl final volume with 4 μl of PROMEGA Green GOTaq 5X Buffer, 0.2 μM primers, 1 mM MgCl2, 5 μl DNA sample, and 1.25 IU Taq DNA polymerase. The cycle conditions were 4 min 94°C, 40 cycles of 30 sec 94°C, 30 sec 58°C, and 20 sec 72°C followed by final elongation of 1 min 72°C. Equine male and female DNA was purified from whole blood (PROMEGA WizardTM Genomic DNA purification kit) and used as positive controls; female and male band patterns were used as reference. Our results showed that sex was confirmed in 4/4 (100%; 3 male and 1 female) of embryos biopsied by micromanipulation (Micro group) and in 5/10 (50%; 1 male and 4 female) of embryos collapsed by manual needle puncture (Needle group). Although the proportion of sex determination in the manually collapsed group is still low, it could be attributed to sample dilution effects. This report provides proof of concept of DNA detection and sex determination in the medium surrounding manually collapsed embryos without blastocoel aspiration. Our findings suggest that this approach to embryo biopsy for sex determination is promising, but more studies are needed to confirm its effectiveness.