49 Collection of Day 7 equine embryos in aluteal cycles in mares

Serial administration of prostaglandin F2a (PGF2) in early diestrus has been recently shown to prevent luteal function in mares and is associated with delayed embryo development in induced aluteal cycles (progesterone deprived environment; <1.0 ng mL-1; Leisinger et al. 2018 Theriogenology 105, 178-183). We hypothesised that embryos collected on Day 7 during induced aluteal cycles would be developmentally delayed compared with those collected from control cycles (progesterone deprived environment but supplemented with exogenous progestogen). Mares were monitored until a preovulatory follicle = 35 mm and the presence of uterine oedema were detected by ultrasonography. Mares were treated IV once with 2000 IU of human chorionic gonadotropin and artificially inseminated every other day with total motile spermatozoa from one stallion of known fertility until ovulation. Mares were examined twice daily to determine the occurrence of ovulation. After ovulation, mares were randomly assigned to the control group (serial PGF2 treatment + long-acting altrenogest) or AL group (serial PGF2 treatment only). Using a protocol to induce aluteal cycles (Leisinger et al. 2018 Theriogenology 105, 178-183), mares in the AL group (n = 7) were treated twice daily with 10 mg of PGF2 (Lutalyse, dinoprost tromethamine, Zoetis, Florham Park, NJ, USA) IM on Days 0, 1, 2, and then once daily on Days 3 and 4. Mares in the control group (n = 4) were treated with serial PGF2 treatment as mentioned before, and treated with a single injection of 225 mg altrenogest (BioRelease Altrenogest LA 150; BET Pharm, Lexington, KY, USA) IM at the time of ovulation. On Day 7 post-ovulation, embryo collection was performed by uterine flushing using lactated Ringer’s solution. The developmental stage of embryos, diameter, and quality were determined using a stereomicroscope and photographed. Embryos were washed 3 times in commercial embryo holding medium (EmCare™ Holding Solution, ICP Bio, Spring Valley, WI) and stained with 1 µg mL-1 of 4’,6-diamidino-2-phenylindole at 38.5°C for 15 min to determine the number of nonviable cells. Data were analysed using t-test or Mann-Whitney U test where appropriate. Statistical significance was set at P = 0.05. Data are reported as mean ± s.e.M. Overall, the developmental stage of control embryos differed from AL embryos (P < 0.03). In the control group, all embryos (n = 4) collected were classified as expanded blastocysts. In contrast, embryos (n = 7) collected from the AL group were all early blastocysts. Furthermore, the mean embryo diameter differed in control v. AL groups, 401 ± 8 mm v. 166 ± 2 mm, respectively (P < 0.001). The quality grades of control embryos (1.3 ± 0.3) were not different from the AL group (1.3 ± 0.2; P > 0.05). All embryos of both groups had less than 10% of blastomeres stained with 4’,6-diamidino-2-phenylindole. This study showed that morphologically normal equine embryos of acceptable quality can be collected on Day 7 in aluteal cycles.