Cross-species multi-omic analyses of great ape ejaculates reveal novel strategies for enhancing sperm cryopreservation in Bornean orangutan (Pongo pygmaeus)

The orangutan (Pongo pygmaeus [Bornean] and Pongo abelii [Sumatran], Linnaeus, 1760) is the most endangered of the great apes, classified as Critically Endangered. Sperm cryopreservation is a valuable tool for banking genetic resources and solving the complexities of relocating animals; however, cryopreservation protocols remain suboptimal for this species. Due to their phylogenetic closeness, this study aimed to explore the applicability of a chimpanzee (Pan troglodytes, Blumenbach, 1775) sperm cryopreservation protocol to orangutans. To guide further modifications to the protocol, we revealed, for the first time, the comprehensive lipidomic and proteomic characterizations of orangutan ejaculates with parallel comparisons to chimpanzee ejaculates. Functional analyses of oxidative and osmotic stress responses were also conducted to provide valuable evidence of the physiological changes and defense mechanisms associated with sperm cryodamage. The cross-species multi-omic analyses revealed that, compared to the chimpanzee, the orangutan sperm lipid profile exhibited significant alterations after the freezing-thawing process, notably characterized by a substantial loss of cholesterol. While interspecies differences in antioxidant enzyme composition and activity were observed, there was insufficient evidence to support a heightened susceptibility of orangutans to oxidative stress. Conversely, orangutan sperm exhibited low tolerance to hypoosmotic conditions. To prevent cryodamage, a modified thawing protocol that implemented a serial dilution approach significantly minimized hypoosmotic shock and improved post-thaw motility to 19%. In conclusion, this study presents the first complete proteome and lipidome analyses of chimpanzee and orangutan ejaculates, providing valuable insights into the physiological changes and defense mechanisms associated with sperm cryopreservation. This knowledge enabled a science-based approach to improving cryopreservation protocols, moving beyond empirical trial-and-error.