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

ABSTRACT: The orangutan (Pongo spp., Linnaeus, 1760) is the most endangered great ape, 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 showed that compared to chimpanzee, the orangutan sperm lipid profile presented significant alterations after the freezing-thawing process, notably showing significant cholesterol loss. While interspecies differences in antioxidant enzyme composition and activity were observed, insufficient evidence supported the orangutan's heightened susceptibility to oxidative stress. Conversely, orangutan sperm exhibited low tolerance to hypoosmotic conditions. To prevent cryodamage, a modified thawing protocol by implementing a serial dilution approach significantly minimized the 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 away from trial-and-error methods.

INSTRUMENT(S):

ORGANISM(S): Pongo Pygmaeus (bornean Orangutan) Pan Troglodytes (chimpanzee)

TISSUE(S): Spermatozoid, Sperm, Seminal Plasma

SUBMITTER: Laura Orama Méar

LAB HEAD: Pei-Shiue Tsai

PROVIDER: PXD073407 | Pride | 2026-03-08

REPOSITORIES: Pride

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			Action	DRS
	20220309_CHIMP_SPERM_1-1.wiff	Wiff
	20220309_CHIMP_SPERM_1-1.wiff.scan	Wiff
	20220309_CHIMP_SPERM_1-2.wiff	Wiff
	20220309_CHIMP_SPERM_1-2.wiff.scan	Wiff
	20220309_CHIMP_SPERM_1-3.wiff	Wiff

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Project description:Background: The cryopreservation of semen is crucial for conserving genetic diversity and reproductive success in endangered species like the Siberian sturgeon. However, this process can cause cryodamage, affecting quality and protein profile of spermatozoa. While cryoprotectants like dimethyl sulfoxide (DMSO) and methanol (MeOH) facilitate post-thaw motility recovery, DMSO-preserved spermatozoa exhibit reduced fertilizing ability. This study investigates how DMSO and MeOH impact the proteome of Siberian sturgeon spermatozoa and examines semen quality parameters. Two complementary approaches of quantitative proteomics, liquid chromatography-mass spectrometry (LC-MS) and two-dimensional difference in gel electrophoresis (2D-DIGE), were used to analyze the proteomic profiles of fresh and cryopreserved spermatozoa, as well as the extracellular medium (EM; n=7 for each group). Results: Cryopreservation led to a decline in motility parameters (MOT, VCL, PROG) and viability, along with an increase in ROS levels, membrane fluidity, and acrosome damage. Despite similar quality parameters between DMSO and MeOH-preserved sperm, DMSO-preserved sperm showed dramatically lower fertilization success (6.2% vs 51.2%). A total of 224 and 118 differentially abundant proteins in spermatozoa cryopreserved with MeOH and DMSO, respectively, were identified compared to fresh samples, with 342 and 363 proteins released into the EM. The most affected proteins by cryopreservation included H2A, CABYR, PYGM, ENO3, DBI and LTA4H. Additionally, 36 and 39 uniquely altered sperm-leakage proteins were identified for MeOH and DMSO cryopreserved samples, respectively. Bioinformatic analysis showed that MeOH-specific proteins were related to chromosomal structure and mitochondrial functionality, while DMSO-specific proteins were mainly involved in acrosome reaction, zona pellucida binding, flagella structure, and nuclear pore organization. These proteins are potentially involved in sturgeon sperm fertilizing ability. The expression of six proteins was verified by western blot analysis. Conclusions: This study provides the first comprehensive proteomic characterization of Siberian sturgeon spermatozoa after cryopreservation with DMSO and MeOH, revealing insights into proteomic changes that affect fertilizing ability and aiding conservation efforts for this endangered species.

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

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