In vitro-produced (IVP) embryos are more vulnerable to cryopreservation than in vivo-derived (IVD) embryos, resulting in lower average rates of conception and live births for vitrified embryos than for non-vitrified embryos [[1], [2], [3]]. This phenomenon primarily occurs due to cryoinjuries experienced by the embryos during the vitrification and warming processes, leading to damage to cells, such as delay in the resumption of protein synthesis, alterations in the expression levels of genes related to pro-inflammatory cytokines, and increased apoptotic activity within the cells [4].

Apoptosis is a highly regulated and programmed process in which immune cells condense cellular contents into small membrane-bound vesicles for clearance [5]. This mechanism is crucial in eliminating potentially harmful or abnormal cells during embryonic development [6]. However, deviations in this balance can contribute to abnormal cell growth, proliferation, and elimination of cells [7]. Cell death is a critical factor controlling mammal embryonic development [[8], [9],]. A notable characteristic of IVP embryos is the generally higher apoptosis rate than their in vivo counterparts, indicating that in vitro conditions are less favorable for embryo development. The number of apoptotic cells is a marker of embryonic quality, influencing embryonic development and cell viability [10]. Apoptosis is regulated by two converging pathways, depending on whether the signals initiating cell death originate within or outside the cell. The extrinsic pathway is primarily activated by tumor necrosis factor (TNF-α) binding to its specific receptor family (TNFR) [11]. The intrinsic pathway is triggered by intracellular or extracellular stress and involves events within the mitochondria [8]. Consequently, pro-apoptotic proteins are released from the intermembrane space into the cytosol [12,13].

Both apoptotic pathways can be activated under specific conditions but are not fully operational in early-stage embryos [14]. The extrinsic apoptotic pathway may be partially inactive in bovine embryos due to a lack of convergence with the intrinsic pathway required to activate effector caspases; this could be attributed to the absence or inactivation of caspase-8 [10]. In bovine embryos, apoptosis may occur through an alternative pathway intensified by the inhibition of caspase-8 and the activation of nuclear factor kappa-B (NF-κB) [15,16]. The signaling pathway involving death receptors is characterized by NF-κB activation, which generally triggers a broad inflammatory response associated with cell survival [17,18]. Bovine blastocysts possess NF-κB inducers such as TNF-α and Interleukin (IL) 1 Beta (IL1β) [19].

NF-κB plays a critical role in regulating the expression of pro-inflammatory cytokines and controlling gene transcription of various inflammatory factors, including TNF-α, IL-6, IL-1β, IL-8, and adhesion molecules [20]. Conversely, the body's natural defense against NF-κB activation involves the production of anti-inflammatory cytokines, such as IL-10 [21]. IL-10 is an important immunoregulatory cytokine produced by various cell types [21]. It has multiple biological functions, including limitation and termination of inflammatory responses, regulation of cellular differentiation, and proliferation of various immune cells [22]. Studies have indicated that IL-10 can inhibit the secretion of TNF-α [19,[22], [23], [24]]. Furthermore, IL-10 can inhibit TNF-α-induced NF-κB activity by blocking the ability of translocated NF-κB to bind to DNA, thereby preventing the expression of pro-inflammatory cytokines [25].

In vitro production and cryopreservation can damage embryonic DNA, triggering the expression of cytokine genes that may hinder post-warming development. IL-10 expression can inhibit NF-κB activity, which is known to upregulate the promoter region of numerous pro-inflammatory cytokine genes. Hence, this study aimed to assess the impact of IL-10 and TNF-α on the competence and cryotolerance of bovine in vitro-produced (IVP) embryos.