Cryopreservation facilitates storage and transportation of germplasm and can be applied to contemporary reproductive techniques such as artificial insemination (AI) [1]. However, cryopreservation of ram semen is much less common than cryopreservation of bull semen, seriously restricting its use. Ram sperm is less resistant to freezing than sperm from many other mammals, due to more unsaturated fatty acids and fewer lipids [2]. Although approximately 40–60% of frozen-thawed sperm are motile [3,4], only 20–30% have biological functions [[5], [6], [7]]. Unfortunately, in current research on cryopreservation, the typical focus is structural protection (plasma membrane, mitochondria, acrosome, etc.) [[8], [9], [10], [11]], with limited or no consideration of biological functions (metabolism, motility, fertilization, etc.). Therefore, characterizing molecular changes in sperm during freezing and thawing should provide a basis for reducing sperm cryo-injury.

Newly synthesized precursor proteins usually need to undergo complex post-translational modifications (PTMs) and processing to become biologically active [12]. However, sperm are highly differentiated cells, generally regarded as lacking transcription and translation. Therefore, sperm mainly regulate their actions through microtubule transport and they rely on PTMs of existing proteins [[13], [14], [15]], making them an ideal cell type for studying protein modifications and functions [16]. Sperm proteins with PTM, e.g., phosphorylation, acetylation, or glycosylation, can be directly involved in sperm-specific functions [17]. Among them, phosphorylation is the most common and important type of covalent modification, with an important role in sperm signal transduction [[18], [19], [20]]. During freezing and thawing, sperm undergo intense changes in osmotic pressures at various stages, including dilution, cooling, and low-temperature equilibration, all of which have the potential to cause physicochemical damage to sperm. Thus, it is particularly important to study dynamic changes in protein phosphorylation during sperm cryopreservation.

Although recent studies have addressed impacts of cryopreservation on the ram sperm proteome (e.g., altering the proteome of ram sperm, affecting sperm quality, and its specific machinery to sustain capacitation and perhaps compromise fertility of post-thaw sperm) [[21], [22], [23]], effects on phosphorylation are not well characterized. We hypothesize that cryopreservation-induced reductions in sperm function are closely related to changes in protein phosphorylation modification. In this study, differentially phosphorylated proteins in ram sperm were analyzed using phosphoproteomics, comparing before and after freezing, to identify potential biological markers of frozen-thawed sperm quality.