Approaches to Improve <i>In Vitro</i> Oocyte Maturation and Somatic Cell Nuclear Transfer Embryo Developmental Potential

Somatic cell nuclear transfer (SCNT), a technique used in agricultural and biomedical fields for cloning livestock and creating animal models of diseases, is very inefficient. Cloned offspring that survive are usually only produced around 1% of the time, but our previous study showed that adding certain immune-system related proteins (FGF2, LIF, and IGF1, known as FLI) during cow egg (oocyte) maturation in a laboratory improved embryo development and cloning efficiency. Additionally, obtaining sheep eggs through a minimally invasive technique called laparoscopic ovum pick-up (LOPU) showed a positive effect on embryo development following in vitro fertilization (IVF). Therefore, we focused on finding the reasons behind improved SCNT efficiency after FLI supplementation as well as the effect of LOPU on sheep cloning. Our first study compared cow oocytes that were treated with FLI during their growth to those that were not. We found that FLI-treated oocytes mature better in two important areas: their DNA (nuclear) and their organelles (cytoplasmic). The treated oocytes went through the first meiotic cell division more smoothly, with fewer problems in the structure that is created to separate DNA (the meiotic spindle). The treated oocytes also had better-organized mitochondria (the cell's energy powerhouses) and cortical granules (the cell's defense mechanism against multiple sperm fertilizing the egg). This suggests that adding FLI helps the oocytes develop more fully and become better candidates for successful cloning or other assisted reproduction techniques. Our second study aimed to understand how adding FLI during oocyte growth in an incubator affects cloned cattle embryos. We analyzed three key areas that are often disrupted during cloning: how the DNA and RNA are "marked" or modified (epigenetic changes), how the cells grow and divide (morphokinetics), and how fats are stored in the embryos. Cloned embryos derived from FLI-treated oocytes had more consistent DNA and RNA modification profiles, increased morphokinetic activity, and enhanced fat accumulation compared to cloned embryos that did not come from oocytes treated with FLI. This suggests that FLI improves embryo development and growth, making the cloning process more successful. In our last study, we looked at how collecting sheep oocytes using a technique called LOPU affects the development of cloned embryos. LOPU-collected oocytes matured better than those from a slaughterhouse, even outside the typical sheep breeding season (short days). During the breeding season, cloned embryos from LOPU oocytes were better at reaching a key development stage (the blastocyst), though the number of cells in the embryos was similar to those made from slaughterhouse oocytes. Sheep that received cloned embryos produced from LOPU oocytes had higher pregnancy rates and fewer losses during pregnancy. Most importantly, using LOPU oocytes increased the success of SCNT cloning from 2% to about 10%, which is a big improvement. This suggests that sheep oocytes collected by LOPU are better at developing into healthy embryos, making the cloning process more successful. In simple terms, this research has helped us understand the different factors that improve the success of cloning in both cows and sheep, making the process more efficient and potentially guiding future research in other species.