57 Influence of donor mare age on pre- and postimplanation embryo development within an equine ovum pick-up-intracytoplasmic sperm injection-embryo transfer (OPU-ICSI-ET) program over a three-year period

In this retrospective study, we examined the influence of donor mare age on the efficiency of an equine ovum pick-up-intracytoplasmic sperm injection-embryo transfer (OPU-ICSI-ET) program during the period from 2018 to 2020. The aim of this study was 2-fold: (1) to investigate the ability of OPU oocytes to develop into viable embryos after fertilisation by ICSI and embryo culture, and (2) to evaluate the pregnancy rate obtained after transfer of the frozen-thawed embryos in recipient mares. To this aim, the OPU-ICSI datasets of 2535 OPU sessions and the ET dataset of 1021 transfers of frozen-thawed embryos were divided in four groups based on the age of the OPU donor mares: less than 9 yr, from 9 to 14 yr, from 15 to 20 yr, over 20 yr. Both datasets were limited to donor mares of warmblood breeds to avoid the breed effect previously reported (Lazzari et al. 2020 J Equine Vet Sci. 89, 103097). Prior to OPU, the donor mares were sedated with detomidine hydrochloride followed by epidural anaesthesia. Follicular aspiration was performed using a 12 G double-lumen needle mounted on a modified native human endovaginal probe. The recovered oocytes were washed in holding medium (H-SOF) and transferred to maturation medium (Dulbecco’s modified Eagle medium (DMEM):F12) supplemented with 10% fetal calf serum, insulin-transferrin-selenium (ITS), LH and FSH for 36 to 38 h. Stallion semen was prepared by gradient centrifugation before piezo-ICSI. Injected oocytes were cultured in mSOF medium for up to 9 days. Embryos were evaluated according to IETS criteria and only grade 1 embryos were frozen in 10% glycerol with slow freezing protocol while retarded and low-quality embryos were discarded. Embryo transfer was performed 4 to 5 days after ovulation. Data shown in Table 1 indicate that preimplantation embryo development was higher (chi-squared test, P < 0.0.05) in donor mares up to 14 yr of age (2.6–2.5 embryos/OPU) than 15–20 yr of age (1.9 embryos/OPU) or more than 20 yr of age (1.0 embryo/OPU). Embryo transfer data indicate no statistical differences among the four groups in pregnancy rate at 12 days after ET (<9 years: 148/202, 72.9%; 9–14 years: 194/272, 71.3%; 15–20 years: 343/456, 75.2%; >20 years: 63/90, 70.0%) and at 50 days of gestation (<9 years: 125/203, 61.6%; 9–14 years:167/272, 61.4%; 15–20 years: 288/456, 63.2%; >20 years: 52/90, 57.8%). In summary, this study indicates that OPU donor mare age influences the rate of preimplantation embryo development, being significantly higher up to 14 yr compared with those of more advanced age. In addition, we found that grade 1 embryos belonging to all four age groups can establish pregnancy at similar rates, suggesting that strict embryo selection before freezing is important for achieving satisfactory pregnancy rate.

Table 1.  Influence of donor mare age on embryo development following OPU-ICSI