A wide variety of Assisted Reproductive Techniques (ART) have been developed to aid in managing equine reproduction to support basic and applied aspects associated with male and female fertility, sperm and oocyte collection and transfer, in vivo and ex vivo embryo production and development, sexing, cloning, and transfer, pregnancy maintenance, pregnancy loss and more as reviewed [[1], [2], [3], [4]]. In humans, preimplantation genetic diagnosis (PGD) was introduced in 1990 [5] to detect and primarily minimize or avoid the inheritance of genetic abnormalities and is now frequently used as an ART to manage and support reproduction programs [[6], [7], [8]].

With the successes in humans, PGD has been adapted for use in other mammalian species such as canine [9], bovine [10], and equine [11] to support reproduction programs. In equine reproduction, PGD has been primarily used for sexing embryos prior to transfer [3,[12], [13], [14], [15], [16]]. Equine embryos produced in vivo, expanded, sexed [12,16], vitrified [17,18], and transferred have resulted in successful pregnancies of the desired sex (73%). These successes are due, in part, to the relatively high level of expertise needed to perform biopsies and collection of embryonic cells (EC) or blastocoel fluid (BF) using costly equipment (e.g., embryo micromanipulator). While relatively successful, the current procedure for sexing equine embryos prior to transfer is restricted to specialized centers and limits the commercial and broader application of under farm or field conditions [13,16].

In place of a specialized embryo micromanipulator to assist in perforating the blastocoel to collect EC and/or BF for sexing equine embryos [[19], [20], [21]] an alternative or practical approach has been studied [22]. Prior to vitrification, a 26g hypodermic needle was used to manually perforate the blastocoel of in vivo produced equine embryos. Although embryos collapsed due to the leakage of BF, they were resilient and recovered with good quality for transfer that resulted in a pregnancy rate of 46% (7/15). While relatively successful and comparable with previously documented results [21,22], the rates are relatively low for a commercial application. Hence, a study is needed to determine if the amount of BF that leaked from perforated embryos would be sufficient to conduct PCR analysis to identify embryo sex and determine quality to support transfer and achieve an acceptable pregnancy rate with the desired sex.

Equine embryo transfer may also involve short-term storage of collected embryos to allow for transport or other events that require time before transfer [[23], [24], [25]], such as sexing of embryos [13]. While there is a multitude of early [[25], [26], [27], [28], [29], [30]] and more contemporary [23,28,31] studies that have addressed various storage techniques of in vivo produced equine embryos, evaluation of storage of in vivo produced embryos subjected to manipulation or perforation, collection of BF, collapse, recovery, and sexing of embryos has apparently not been documented.

The present study evaluated the use of in vivo produced equine embryos collected 8d post-ovulation that were subjected to manual perforation using a hand-held, small-gauged needle and PCR analysis of the leaked blastocoel fluid (BF) for sexing embryos. Experiment 1: Determine embryo sex using BF and DNA amplification of the TSP-Y and AMEL-X and -Y genes. Experiment 2: Determine embryo sex as in Experiment 1 and evaluate re-expansion and quality of collapsed embryos 24h after storage at 25 °C (room temperature) or 5 °C (cold temperature).