Semen cryopreservation results in the differential remodeling of the molecules presented in sperm, and these alterations related to reductions in sperm quality and its physiological function have not been fully understood. Given this, this study aimed to investigate the cryoinjury mechanism of goat sperm by analyzing changes of the metabolic characteristics in sperm during the cryopreservation process. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique was performed to explore metabolite profiles of fresh sperm (C group), equilibrated sperm (E group), and frozen-thawed sperm (F group). In total, 2570 metabolites in positive mode and 2306 metabolites in negative mode were identified, respectively. After comparative analyses among these three groups, 374 differentially abundant metabolites (DAMs) in C vs. E, 291 DAMs in C vs. F, and 189 DAMs in E vs. F were obtained in the positive mode; concurrently, 530 DAMs in C vs. E, 405 DAMs in C vs. F, and 193 DAMs in E vs. F were obtained in the negative mode, respectively. The DAMs were significantly enriched in various metabolic pathways, including 31 pathways in C vs. E, 25 pathways in C vs. F, and 28 pathways in E vs. F, respectively. Among them, 65 DAMs and 25 significantly enriched pathways across the three comparisons were discovered, which may be tightly associated with sperm characteristics and function. Particularly, the functional terms such as TCA cycle, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, glycine, serine and threonine metabolism, alpha-linolenic acid metabolism, and pyruvate metabolism, as well as associated pivotal metabolites like ceramide, betaine, choline, fumaric acid, L-malic acid and L-lactic acid, were focused on. In conclusion, our research characterizes the composition of metabolites in goat sperm and their alterations induced by the cryopreservation process, offering a critical foundation for further exploring the molecular mechanisms of metabolism influencing the quality and freezing tolerance of goat sperm. Additionally, the impacts of equilibration at low temperature on sperm quality may need more attentions as compared to the freezing and thawing process.

This comprehensive review critically examines the application of proteomics in understanding sperm cryoinjury mechanisms in livestock animals, in the context of the widespread use of semen cryopreservation for genetic conservation. Despite its global adoption, cryopreservation often detrimentally affects sperm quality and fertility due to cryoinjuries. These injuries primarily arise from ice crystal formation, osmotic shifts, oxidative stress, and the reorganization of membrane proteins and lipids during freezing and thawing, leading to premature capacitation-like changes. Moreover, the cryopreservation process induces proteome remodeling in mammalian sperm. Although there have been technological advances in semen cryopreservation, the precise mechanisms of mammalian sperm cryoinjury remain elusive. This review offers an in-depth exploration of how recent advancements in proteomic technologies have enabled a detailed investigation into these molecular disruptions. It presents an analysis of protein-level alterations post-thaw and their impact on sperm viability and functionality. Additionally, it discusses the role of proteomics in refining cryopreservation techniques to mitigate cryoinjury and enhance reproductive outcomes in livestock. This work synthesizes current knowledge, highlights gaps, and suggests directions for future research in animal reproductive science and biotechnology.

The changes in semen and cryodamage after the cryopreservation process negatively affect sperm function and motility. However, possible proteomic alterations of yak semen during cryopreservation have not yet been achieved. In this study, we compared proteomes of fresh and frozen thawed yak sperm using iTRAQ combined with LC-MS/MS proteome approach. Totally, 2064 proteins were quantitatively identified, including 161 in fresh sperm that showed significant differences compared to frozen thawed sperm. According to the Gene ontology (GO) enrichment analysis, differentially expressed proteins (DEPs) are predominantly associated with spermatogenesis, tricarboxylic acid cycle, ATP synthesis, and differentiation biological process. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPs were mainly involved in metabolic pathways related to pyruvate metabolism, carbon metabolism, glycolysis/gluconeogenesis, together with the citrate (TCA) cycle. In the analysis of the protein-protein interaction (PPI) network, 15 potential proteins (PDHB, DLAT, PDHA2, PGK1, TP5C1, etc.) that could be related to the sperm quality of the yaks were obtained. Furthermore, 6 DEPs were validated by parallel reaction monitoring (PRM), confirming that the iTRAQ data were reliable. These results indicate that cryopreservation alters the proteome of yak sperm, which is possibly related to cryodamage and fertilization ability.

The change of sperm protein profile after the cryopreservation process may influence fertilization and early embryonic development. The purpose of the present study was to identify ram sperm proteomic modifications induced by the cryopreservation process using the isobaric tags for relative and absolute quantification labeling technology (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. Semen samples were collected from five Yunnan semi-fine wool rams using an electroejaculator. Sperm motility (CASA), plasma membrane (HOST test), and acrosome integrity (FITC-PSA) were evaluated after freeze-thawing. The total proteins of fresh and frozen-thawed sperm were extracted and purified, followed by identifying ram sperm proteomic modifications using the isobaric tags for relative and absolute quantification labeling technique (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. The results showed a significant reduction (P < 0.05) in all sperm parameters after thawing. 126 differentially abundant proteins (DAPs) were identified through comparison of the proteomes between fresh and frozen-thawed sperm. Among them, 90 proteins were down-regulated after the cryopreservation process. The remaining 36 proteins were up-regulated in frozen-thawed sperm. The results of functional annotation demonstrated the potential relationship of 10 DAPs with oxidoreductase activity. 18 and 15 DAPs may be involved in the stress and carbohydrate metabolic process, respectively. Furthermore, 8 DAPs may be functionally associated with reproduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results demonstrated the primary enrichment of these identified DAPs in metabolic activities, disease, and oxidative phosphorylation. In order to confirm the reliability of the iTRAQ results, the changing trends of 10 proteins analyzed by PRM were similar to those of the corresponding proteins identified by iTRAQ. In conclusion, the cryopreservation process modifies the proteome of ram sperm, possibly leading to compromised fertility of post-thaw sperm. Additionally, the identified DAPs in this study may function as potential biomarkers for assessing the post-thaw quality of ram semen.

The aim of this study is to do a study of cryoinjury and ischaemic injury on testicular graft during cryopreservation and transplantation. According to time at 1, 3, 7 and 14 days after transplantation, the grafts were collected for immunohistochemistry assay for CD34 (blood vessel marker), VEGF (neoangiogenesis marker), caspase-3 (apoptosis marker) MAGE-A4 (germ cell marker). A significant increase was observed in the density of VEGF-positive blood vessels on day 3, reached a peak on day 7. On post-transplant day 3, a sharp increase occurred in the rate of spermatogonia-expressing caspase-3 until the day 7. At 14th day after transplantation, the spermatogonia number per round tubule of nonfrozen grafts was 41 ± 5.9% from that of fresh control tissues, while, in frozen-thawed grafts, the spermatogonia number per round tubule was 36.8 ± 4.6% from that of fresh control tissues. In testicular grafts, angiogenesis initiated reperfusion from day 3, and the formation of new blood vessel generally is completed about 7 days after transplantation. Angiogenesis in grafts after transplantation plays a crucial role in the restoration of function. Therefore, minimising ischaemic injury as well as improvement of cryopreservation protocols are needed to improve testicular graft after freezing, thawing and grafting.

In this study, we developed an optimal cryopreservation procedure for Varicorhinus barbatulus sperm. To this end, we optimized (1) the types and dilution ratios of extenders; (2) types and final concentration of cryoprotectants; and (3) freezing conditions, including equilibration time, height above the surface of liquid nitrogen (LN), and the cooling times in the two-step cooling method. The optimum result was obtained when the sperm was diluted at a 1:9 ratio in D-17 with 10% methanol, equilibrated at 4 °C for 10 min, held at 7 cm above LN for 2 min, and finally stored in LN. After storage for 12 h in LN, the sperm was thawed in a water bath at 40 °C for 6s, the post-thaw sperm motility was 66.10 ± 7.12%, while the corresponding rate for fresh sperm was 87.08 ± 2.38%. Using computer-assisted sperm analysis, we found a significant decrease in the motility parameters of post-thaw sperm, especially the parameters related to velocity. To evaluate the effects of cryopreservation on the structural integrity of sperm, transmission electron microscopy and scanning electron microscopy were employed, which showed the defects in frozen sperm, including: abnormal heads, damaged plasma membranes, broken tails, and the disappearance of the mitochondrial internal crest. In addition, we determined the mitochondrial membrane potential to assess the functional integrity of frozen sperm. Our results showed a decrease in the mitochondrial function of frozen sperm. This procedure could be used alongside cryopreservation of V. barbatulus and supports its commercial-scale production and species conservation.

Neutrophils are the first line defenders in the innate immune response, and rapidly migrate to an infected or injured area. Recently, bidirectional migration of neutrophils to the wound and the corresponding functions have become popular research pursuits. In zebrafish larvae, CXCR1/CXCL8 is the predominant chemoattractant pathway to recruit neutrophil to wound, while CXCR2/CXCL8 pathway mediate neutrophil dispersal in wound after injury. Here, we found that both CXCR1/CXCL8 and LTB4/BLT1 signals are activated in zebrafish heart after cryoinjury. And with a CXCR1/2 selective inhibitor (SB225002) treatment, the recruitment of neutrophils was not affected, but reverse migration of neutrophils was inhibited after cryoinjury of heart. We suggested that the neutrophil recruitment to cryoinjured area might be mediated by LTB4/BLT1 signals at the presence of SB225002. Therefore, SB225002 treatment resulted more accumulation and long retention of neutrophils in the injured heart. The long retention of neutrophils in the wound promoted revascularization in the injured heart; however, the AKT/mTOR pathway was inhibited and the regeneration was impaired. Our findings suggest that retention of neutrophils is a well-orchestrated process and might regulate regeneration by the AKT/mTOR pathway.

Post-thawed sperm quality parameters vary across different species after cryopreservation. To date, the molecular mechanism of sperm cryoinjury, freeze-tolerance and other influential factors are largely unknown. In this study, significantly dysregulated microRNAs (miRNAs) and mRNAs in boar and giant panda sperm with different cryo-resistance capacity were evaluated. From the result of miRNA profile of fresh and frozen-thawed giant panda sperm, a total of 899 mature, novel miRNAs were identified, and 284 miRNAs were found to be significantly dysregulated (195 up-regulated and 89 down-regulated). Combined analysis of miRNA profiling of giant panda sperm and our previously published data on boar sperm, 46, 21 and 4 differentially expressed (DE) mRNAs in boar sperm were believed to be related to apoptosis, glycolysis and oxidative phosphorylation, respectively. Meanwhile, 87, 17 and 7 DE mRNAs in giant panda were associated with apoptosis, glycolysis and oxidative phosphorylation, respectively. Gene ontology (GO) analysis of the targets of DE miRNAs showed that they were mainly distributed on membrane related pathway in giant panda sperm, while cell components and cell processes were tied to the targets of DE miRNAs in boar sperm. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DE mRNAs indicated that most of these DE mRNAs were distributed in membrane signal transduction-related pathways in giant panda sperm, while those in boar sperm were mainly distributed in the cytokine-cytokine receptor interaction pathway and inflammatory related pathways. In conclusion, although the different freezing extenders and programs were used, the DE miRNAs and mRNAs involved in apoptosis, energy metabolism, olfactory transduction pathway, inflammatory response and cytokine-cytokine interactions, could be the possible molecular mechanism of sperm cryoinjury and freeze tolerance.

OBJECTIVE: To investigate the long-term functional change of cryoinjury-induced detrusor underactivity (DU) and the therapeutic potential of repeated low-energy shock wave therapy (LESW).
METHODS: Fifty-six female Sprague-Dawley rats were assigned into sham and cryoinjury of bladder with or without LESW (0.05 or 0.12 mJ/mm2; 200 pulses; twice a week for 2 weeks after cryoinjury). Under halothane anesthesia, an incision was made in lower abdomen, and cryoinjury was provoked by bilateral placement of a chilled aluminum rod on the bladder filled with 1 ml saline. Measurement of contractile responses to KCl and carbachol in vitro, conscious voiding, and histological and protein changes were performed on week 1, 2, and 4 after cryoinjury.
RESULTS: Cryoinjury of bladder induced a significant decrease in the detrusor contraction amplitude at week 1 (55.0%) and week 2 (57.2%), but the decrease in the contractile response to KCl and carbachol was only noted at week 1. At week 1, significantly increased COX-2 and TGF-β1 expression accompanied a decrease of VEGF and CGRP expression. At week 4, there was a partial recovery of voiding function and a significant increase in the Ki-67 staining. LESW treatment at higher energy level further amplified the Ki-67 staining and improved the recovery of contraction amplitude and the expression of TGF-β1 and VEGF.
CONCLUSIONS: Cryoinjury of detrusor induces DU/UAB with functional impairment lasting for up to 4 weeks, but the associated molecular changes are restored by 2 weeks. LESW improved bladder wall composition, and hastened functional recovery from cryoinjury.

Cryoprotectants (CPAs) are critical to successful cryopreservation because they can protect cells from cryoinjuries. Because of the limitations of current CPAs, especially the toxicity, the search for new effective CPAs is attracting increasing attention. In this work, we reported that natural biocompatible osmoprotectants, which could protect cells from osmotic injury in various biological systems, might also be ideal candidates for CPAs. Three representative biocompatible osmoprotectants (proline, glycine, and taurine) were tested and compared. It was found that, aside from presenting a different ability to prevent osmotic injury, these biocompatible osmoprotectants also possessed a different ability to inhibit ice formation and thus mitigate intra-/extracellular ice injury. Because of the strongest ability to prevent the two types of injuries, we found that proline performed the best in cryopreserving five different types of cells. Moreover, the natural osmoprotectants are intrinsically biocompatible with the cells, superior to the current state-of-the-art CPA, dimethyl sulfoxide (DMSO), which is a toxic organic solvent. This work opens a new window of opportunity for DMSO-free cryopreservation, and sheds light on the applications of osmoprotectants in cryoprotection, which may revolutionize the current cryopreservation technologies.