Since their establishment, the in vitro maturation (IVM) systems for oocytes have been extensively utilized in domestic animals, including pigs, cows, and sheep [[1], [2], [3]]. However, many problems, such as low maturation rates and subsequent low developmental abilities of embryos, remain to be addressed. These problems mainly result from the considerable differences between in vitro and in vivo oocyte maturation, such as the variations in the microenvironment, fluid mechanics, nutrients, and cell junctions [4]. In addition, factors, such as light exposure and high oxygen concentrations, easily affect during in vitro culture.

Researchers have exerted great efforts, such as by adding growth factors, hormones, and antioxidants to the culture medium, to enhance the maturation quality of porcine oocytes [5]. However, these substances are insufficient for effective quality improvement of porcine oocyte IVM in an artificial environment. The inefficient IVM of oocytes is primarily attributed to the complex 3D cell arrangements in vivo, which involve intricate cell–cell and cell–substrate interactions and complex nutrient delivery through gap-junction intercellular communication (GJIC). The replication of these factors in conventional culture systems presents a challenge [6]. Therefore, further optimization is required for the IVM technology system for porcine oocytes.

A 3D culture is a novel cell culture technique that has achieved tremendous progress. Researchers initially observed that alginate hydrogels can be used as an in vitro culture substrate for various organs and embryos [7,8]. Moreover, alginate-hydrogel embedding can effectively promote in vitro follicular development in mice [9,10] and nonhuman primates [11]. Researchers have shown that alginate hydrogel benefits the in vitro elongation of porcine embryos [7] and enhances the development of oocytes detached from cumulus cells in cats [12]. In addition, Matrigel combined with low-attachment plates substantially prolonged in vitro development in the late blastocyst stage, which is critical for the identification of molecular and morphological changes during human embryogenesis [13]. Gorczyca G et al. observed that encapsulated porcine oocytes in fibrin–alginate beads can maintain the 3D organization of cumulus–oocyte complexes (COCs) for 96 h [14]. However, they neither further assessed the developmental potential of oocytes culture via the 3D method nor optimized the fibrin–alginate embedding method. Therefore, a suitable 3D culture method must be established for porcine oocytes, and the mechanism of 3D culture method in influencing the IVM of porcine oocyte must be investigated.

The main aim of this study were to investigate the effects of three different 3D culture methods on the in vitro culture of porcine oocytes and to establish an alginate hydrogel embedding system that benefits the IVM of porcine oocytes.