13 Donor age effects on embryo morphokinetics in Holstein cattle: insights into prepubertal embryo development

Genomic-based embryo selection has intensified the race to produce genetically superior animals, leading to a surge in demand for assisted reproductive technologies such as ovum pickup (OPU) and in vitro embryo production. These techniques enable offspring generation from very young animals, well before sexual maturity. Although these prepubertal breeding methods have been commercially adopted, particularly for Holstein cattle, challenges persist in the successful fertilization, development, and implantation of embryos from prepubertal donors. This study aimed to evaluate embryo morphokinetic activity across different donor ages to identify age-related differences in preimplantation embryo development in Holstein dairy cattle. OPU was performed on 253 Holstein donors, leading to a total of 1091 embryos produced in vitro. Embryos were developed at a commercial IVF laboratory and shipped fresh to a Holstein breeding facility. Upon arrival, the Day 7 embryos were evaluated, and a 30-s video was captured of each embryo with a View 4K Camera mounted to a Nikon SMZ 1270 microscope. Embryos were subsequently transferred fresh into eligible recipients. Pregnancy was confirmed by ultrasound at 30, 60, and 180 days after transfer. Donors were grouped by age in months at date of OPU. Videos of embryos were processed with graphic image processing techniques to quantify real-time embryo morphokinetic activity, which is known to be an indicator of embryo viability, stress, and metabolic activity. Embryo morphokinetic activity was grouped based on donor age. Morphokinetic activity results were analyzed with ANOVA with significance set at P < 0.05. Donor ages ranged from 6 to over 20 months. Younger donors had lower morphokinetic activity (0.4 at 6–7 months) than older donors (0.97 at >20 months, P < 0.001). Overall pregnancy rate across all donor age groups was 43%, with embryos from young 6- to 7-month-old donors achieving 49% and 46% pregnancy outcomes, respectively. Embryos from 8-month-old donors displayed a 142% increase in activity and decreased pregnancy outcomes (P < 0.001). Embryo activity from donors over 12 months in age remained elevated, although pregnancy outcomes were variable. This study revealed critical findings to embryo developmental activity based on donor age, which is likely linked to puberty and hormone production. As Holstein heifers are expected to reach puberty at ~9–12 months of age, it was interesting to find that the lowest embryo activity was observed from prepubertal donors and embryo activity underwent rapid increases and fluctuations during the initial onset of puberty (8 months). These findings emphasize the relationship between donor age and embryo morphokinetic activity in Holstein cattle, noting significant changes around puberty. Further research will aim to capture earlier developmental stages to better understand the impact of donor age on embryo development and refine prepubertal donor management to enhance the efficacy of genomic-based embryo selection.