Simplified Genetic Tests for Equine Embryos During a Standard Embryo Transfer

The goals of Experiment 1.a (Exp.1a) are to confirm the concept of secretion/leakage of genetic material from the D7 embryos into the surrounding media and to compare the concentration of DNA retrieved when using two different media: commercial embryo holding solution (EHS) and tissue culture medium. In our preliminary experiments, we used tissue culture media, however EHS is the preferred media used for holding and shipping embryos, therefore EHS would be advantageous. Mares will be inseminated with fresh semen and D7 embryos will be collected by routine embryo collection procedure, using LRS [2]. The recovered embryos will be washed 10 times with LRS, measured using an eyepiece micrometer, and graded based on morphology [1]. The washing solution from the last two washing wells will be preserved by adding RNAlater (1:1 volume). Embryos (n=12) will be divided into two groups. Embryos in Group 1 (n=6) will be placed in 2mL of EHS, (Syngro®, Vetoquinol) at 37°C for 24 hours in non-embryo toxic plastic tubes, as typically performed when packaging an embryo for shipment [1]. Embryos in Group 2 (n=6) will be placed in a tissue culture medium as described in the preliminary experiment (in 100 uL of equilibrated DMEM/F-12 medium with 10% FBS and 25 ìg/mL gentamycin under the oil in a mixed gas incubator (5% O2, and 5% CO2 at 37°C for 24 hours). Experiment 1.b. The goal of Experiment 1.b (Exp. 1.b) is to determine the concentration of DNA present in the holding media under routine embryo-holding conditions. Holding and shipping equine embryos for up to 24 hours at 4°C or up to 12 hours at room temperature (22-25°C) has become common practice in the equine industry. Numerous studies have demonstrated that this temporary storage does not lower pregnancy rates when compared to fresh embryos that are transferred immediately after collection [1, 5]. Thus, we will test these two conditions and determine the amount of DNA in spent media. In this experiment, frozen semen from either HYPP or HERDA carrier stallions (heterozygous stallions) will be used for insemination, breeding half of the mares with the HYPP-affected stallion (Group A) and the other half with the stallion carrier of HERDA (Group B) until we collect 14 embryos from each stallion. This will result in approximately 50% heterozygous embryos and 50% homozygous for each mutation, which will help ensure that this technique can effectively detect the mutation. As in Exp. 1.a, embryos will be collected, washed, placed in 2mL of EHS (Syngro®), similar to the routine embryo packaging for shipment [1]. Embryos from each group will be randomly divided into two subgroups; half of the embryos in Group A (HYPP-embryos) will be held at room temperature (22-25°C) for 12 hours (Group A.1 n=7)), and the remaining HYPP-embryos will be placed in a passive cooling device (Equitainer®, Hamilton Thorne Research Inc.) for 24 hours at 4°C (Group A.2 n=7). We will subdivide the embryos in group B (HERDA-embryos) the same way with half of the embryos in Group B (HERDA) will be held at room temperature (22-25°C) for 12 hours (Group B.1 n=7)), and the remaining HYPP-embryos will be placed in a passive cooling device (Equitainer®, Hamilton Thorne Research Inc.) for 24 hours at 4°C (Group B.2 n=7).