As the dominant livestock on the Qinghai-Tibet Plateau, Tibetan sheep (Ovis aries) exhibit critically low reproductive capacity under harsh plateau conditions [1]. In vitro embryo production (IVP) technology in sheep, combining ovum pick-up (OPU) with systems for in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC), has enabled commercial genetic improvement and conservation of endangered breeds. Each OPU session yields 10–14 oocytes, resulting in the production of 3–5 transferable embryos, with subsequent pregnancy rates exceeding 50 % following embryo transfer [2,5]. However, its efficiency remains constrained by suboptimal blastocyst development rates, heterogeneity in oocyte quality, and developmental abnormalities potentially associated with prolonged in vitro culture [3,4]. Therefore, enhancing the in vitro production efficiency of their embryos is pivotal for sustainable development of high-altitude animal husbandry [4]. In vitro oocyte maturation in the laboratory plays a pivotal role in the production of embryos, however research shows that oocytes matured in vivo exhibit superior quality, likely due to the more favorable microenvironment [5]. Follicular fluid is the essential component of the oocyte microenvironment, and studies have shown that adding a small amount of sheep follicular fluid during the maturation process can enhance oocyte development and maturation [6,7]. Similarly, bovine follicular fluid promotes the expansion of cumulus cells and oocyte maturation [8]. Interestingly, guinea pig follicular fluid can improve the maturation rate of bovine oocytes, which indicates that the role of follicular fluid is conserved across different species [9], but the mechanism of action is not clear.

Myo-inositol (MYO) an active agent in follicular fluid, plays a multifaceted role in reproductive biology [10]. Supplementation of bovine embryo culture medium with 0.04 g/L MYO was shown to significantly enhance morula and blastocyst formation, thereby improving the in vitro developmental competence of bovine embryos [11]. Additional studies have revealed that MYO also enhanced the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos [12]. However, although integrated ultrasensitive metabolomics and single-cell transcriptomics showed that inositol was a key regulator in ovine follicular fluid, the effect of myo-inositol on ovine oocyte maturation was not explored [12].

This study delves into the effects of myo-inositol on the in vitro maturation of Tibetan sheep oocytes and embryonic development by incorporating it into the culture medium. The quality of oocytes was evaluated through various metrics, including ROS levels, GSH concentration, mitochondrial activity, mitochondrial membrane potential, calcium ion concentration, and cortical granule distribution. By shedding light on the role of myo-inositol in this context, this study aims to lay the groundwork for refining the in vitro oocyte maturation system and enhancing the efficiency of in vitro embryo production in Tibetan sheep.