22 Lowered morphokinetic activity in early somatic cell nuclear transfer embryos derived from cytokine-supplemented oocytes

Somatic cell nuclear transfer (SCNT) is an important agricultural and biomedical research tool, but it remains highly inefficient. The addition of FGF-2, LIF, and IGF-1 (FLI) in bovine maturation medium (BMM) improved SCNT efficiency by increasing blastocyst development and pregnancy rates (Keim et al. 2023 Reprod. Fertil. Dev. 35, 575–588). It has not been reported how FLI supplementation during oocyte maturation affects subsequent embryonic health throughout in vitro culture. Recently, lower morphokinetic activity was correlated with increased hatching and development after IVF and embryo transfer (Wells et al. 2022 Dairy 3, 849–861). Thus, this study aimed to evaluate real-time morphokinetic activity of BMM-derived SCNT, FLI-derived SCNT, and IVF embryos at three stages: 2-cell, 8-cell, and blastocyst. Bovine oocytes were matured for 21 h at 38.5°C in either FLI-supplemented or standard BMM. Half of the BMM-matured oocytes underwent our IVF protocol (Keim et al. 2023). The remaining MII oocytes from BMM and FLI were used in our SCNT protocol (Kaim et al. 2023). Resulting embryos were placed in a well-of-the-well system to allow individual identification. During the first 48 h, 2-cell and 8-cell embryos were recorded. On Day 7, blastocysts were recorded on a Nikon inverted microscope at ×200 for 30 s. Only embryos recorded at all three developmental stages were sent to EmGenisys for analysis. EmGenisys software subtracts the pixels of each frame from the previous one to determine the morphokinetic activity of the embryo in real time. All groups had four replicates. In Jamovi, a generalized linear model was used to assess the difference in morphokinetic activity within and between the treatment groups. Statistical differences were considered significant when P < 0.05. In total, 29 embryos (FLI, 12; BMM, 7; IVF, 10) were evaluated. Blastocyst rates were 50% (FLI), 36.1% (BMM), and 35.7% (IVF). Regardless of the treatment group, all embryos were less active at the 2-cell stage than at the 8-cell stage (P < 0.001) and less active than blastocysts (P < 0.001). FLI 8-cell embryos were less active than blastocysts (0.410 vs. 0.529; P < 0.001), whereas no difference was observed between BMM 8-cell embryos and blastocysts. IVF 8-cell embryos were more active than blastocysts (0.532 vs. 0.492; P < 0.05). When morphokinetic activity was assessed at each stage, FLI embryos moved less at the 2-cell and 8-cell stages than either BMM embryos (0.304 vs. 0.402; 0.410 vs. 0.564; P < 0.001) or IVF embryos (0.304 vs. 0.379; 0.410 vs. 0.532; P < 0.001). BMM embryos had more activity at the blastocyst stage than IVF embryos (0.560 vs. 0.492; P < 0.05). No difference was observed between FLI and IVF blastocysts. Embryo morphokinetic activity has previously been linked to metabolic activity in cows (Yaacobi-Artzi et al. 2022 PLoS One 17, e0276642), mice (Wolff et al. 2013 Hum. Reprod. 28, 1776–1782), and humans (Ferrick et al. 2020 Hum. Reprod. 35, 2004–2016). Thus, FLI-derived SCNT embryos are less metabolically active during early development, suggesting that FLI supplementation in BMM serves as protection from environmental stress.