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Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca 2+ -binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been previously investigated. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit semen (spermatozoa and extracellular fluid), to examine the effects of freeze-thawing on proteins changes in semen. Comparative analysis and identification of proteins was carried out using 2-dimensional difference in-gel electrophoresis coupled with a matrix-assisted laser desorption/ionization mass spectrometry. Proteomic raw data are available via ProteomeXchange with identifier PXD034832 for spermatozoa and PXD034853 for extracellular fluid. Respectively, 107 and 28 proteins differed in abundance in spermatozoa and extracellular fluid between fresh and frozen groups. Most of these proteins were involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability. These results broaden the understanding of the effects of cryopreservation on rabbit semen and represent a new starting point for the development of improved freezing procedures., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)