Search Results (26)

Ark shells are a group of bivalves that exhibit extraordinary adaptability to the dual environmental pressures of low oxygen and osmotic imbalance. These challenges are particularly pronounced in intertidal zones, where organisms are subjected to rapid and drastic changes in their surroundings. This research investigated the molecular mechanisms that underpin their survival and adaptive strategies, with particular focused on sodium–potassium ATPase (NKA), a pivotal enzyme responsible for maintaining cellular ion transmembrane gradients and ensuring cellular homeostasis under stress conditions. By utilizing genome assemblies and transcriptomics datasets from multiple ark shell species, we successfully identified two distinct NKA-α subunits and two NKA-β subunits, which are essential components of the NKA complex. Moreover, the discovery of a conserved NKA-interacting protein (NKAIN) highlights the complexity and evolutionary significance of the NKA-NKAIN system in ark shells. Phylogenetic analysis revealed a high degree of conservation in the NKA-α and NKA-β subunits across ark shells, suggesting strong selective pressures to preserve their functionality. However, the marked divergence observed between the two NKA-β subunits suggests that they may serve distinct roles in ion transport, potentially specialized for specific environmental conditions or stress responses. Comparative transcriptomic analysis further revealed the regulatory roles of NKA and NKAIN in the adaptive responses to hypoxia and osmotic stress, showing that these genes are dynamically modulated at the transcriptional level in response to environmental challenges. These findings provide a molecular foundation for understanding the osmotic adaptation mechanisms in ark shells and offer novel insights into their ability to thrive in mudflat habitats. This comprehensive exploration of the NKA-NKAIN system not only enhances our understanding of the resilience of ark shells but also provides valuable insights into the molecular and physiological strategies employed by bivalves in intertidal environments. Full article

Background: Abnormal sodium-dependent hexose reabsorption in the proximal tubule, accompanied by a functional decrease in sodium and water reabsorption under conditions of increased volemia, may be attributed to a dysfunction of primary transporters related to a genetic defect in the Na,K-ATPase gamma subunit. Methods: We examined two sisters, aged 6 and 8 years, who presented with hypercalciuria, glucosuria, fructosuria, galactosuria, and atypical proteinuria. Primary diabetes, galactosemia, and fructosemia were excluded, suggesting a defect in cellular hexose transport in the proximal tubule. We conducted tests on the family members to assess the impact of gradually increasing volemia, using a water-loading test, on tubular H⁺ transport and urinary excretion of calcium, citrate, endothelin-1 (ET-1), and atypical proteins. Whole-exome sequencing was performed in the affected patients to identify the genetic basis of this phenotype. Results: Extended investigations revealed a complex defect in tubular H⁺ transport, calcium and citrate handling, and atypical proteinuria, resulting from water load-driven overproduction of endothelin-1 (ET-1). Genetic analysis identified a heterozygous pathogenic variant, c.80G>A, p.(Arg27His), in the FXYD2 gene, which encodes the gamma subunit of sodium/potassium-transporting ATPase. Conclusions: Our findings provide evidence that a defect in FXYD2 (splice form a) leads to functional impairment of proximal tubular hexose reabsorption. This is the first report on the metabolic consequences of a pathogenic FXYD2 variant affecting the gamma subunit of sodium/potassium-transporting ATPase in humans. The genotype–phenotype correlation in two siblings with a sodium-dependent defect in fructose, galactose, and glucose renal reabsorption allowed us to characterize a disease with a distinct clinical course and biochemical profile, not previously reported in the medical literature or genetic databases. Analysis of this condition was crucial for the early introduction of reno-protective treatment aimed at slowing the progression of nephropathy and for risk assessment in family members, which was essential for genetic counseling. Full article

A surge has been observed in the use of pesticides to boost agricultural yield in order to feed the continuously increasing human population. Different types and classes of broad-spectrum insecticides are in use, and the number is constantly increasing with the introduction of new ones. Keeping in view the broad-spectrum effects of organophosphate pesticides including Malathion (MLN), their use is continuously increasing without appraising their toxic impacts on non-target organisms. The continuous rise in the use of MLN has led to its presence, persistence, and transport to water bodies globally, subsequently affecting commercially valuable aquatic organisms. The current study was conducted to assess MLN-induced hematological and biochemical toxicities in the brain of a commercially valuable indigenous major carp, rohu, Labeo rohita. The fish was exposed to an acute concentration of commercial-grade MLN. The LC₅₀ of MLN (5 µg/L) led to behavioral inconsistencies and subtle impacts on the hematology (an increase in white blood cells and a reduction in red blood cells, hemoglobin, packed cell volume level, and mean corpuscular hemoglobin concentration) and biochemistry (an increase in reactive oxygen species, lipid peroxidation, activities of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, glutathione, glutathione reductase, glutathione peroxidase, and glutathione-S-transferase) but a reduction in total protein content and activity of Na⁺/K⁺ ATPases) in the brain tissues. MLN also inhibited the activity of Acetylcholinesterase, while it led to an increase in Acetylcholine. Significant changes were observed in the serum biochemical profile; for example, glucose, cholesterol, potassium, urea, and total bilirubin increased, whereas total protein, sodium, chloride, albumin, and inorganic phosphate decreased after exposure. The current study clearly classified MLN as highly toxic to rohu. Therefore, the extra-judicious use of MLN should be strictly supervised. Studies concerning the real-world concentration of pesticides should be carried out on regular basis to mitigate the echoing issue of pesticide-based pollution. Full article

Multidrug-resistant tuberculosis (MDR-TB) patients are treated with a standardised, short World Health Organization (WHO) regimen which includes clofazimine (CFZ) and bedaquiline (BDQ) antibiotics. These two antibiotics lead to the development of QT prolongation in patients, inhibiting potassium (K⁺) uptake by targeting the voltage-gated K⁺ (Kv)11.1 (hERG) channel of the cardiomyocytes (CMs). However, the involvement of these antibiotics to regulate other K⁺ transporters of the CMs, as potential mechanisms of QT prolongation, has not been explored. This study determined the effects of CFZ and BDQ on sodium, potassium–adenosine triphosphatase (Na⁺,K⁺-ATPase) activity of CMs using rat cardiomyocytes (RCMs). These cells were treated with varying concentrations of CFZ and BDQ individually and in combination (1.25–5 mg/L). Thereafter, Na⁺,K⁺-ATPase activity was determined, followed by intracellular adenosine triphosphate (ATP) quantification and cellular viability determination. Furthermore, molecular docking of antibiotics with Na⁺,K⁺-ATPase was determined. Both antibiotics demonstrated dose–response inhibition of Na⁺,K⁺-ATPase activity of the RCMs. The greatest inhibition was demonstrated by combinations of CFZ and BDQ, followed by BDQ alone and, lastly, CFZ. Neither antibiotic, either individually or in combination, demonstrated cytotoxicity. Molecular docking revealed an interaction of both antibiotics with Na⁺,K⁺-ATPase, with BDQ showing higher protein-binding affinity than CFZ. The inhibitory effects of CFZ and BDQ, individually and in combination, on the activity of Na⁺,K⁺-ATPase pump of the RCMs highlight the existence of additional mechanisms of QT prolongation by these antibiotics. Full article

ATP-binding cassette (ABC) transporters, particularly those of subfamily B, are involved in cell detoxification, multidrug resistance, drug treatment pharmacodynamics, and also ecological adaptation. In this regard, ABCB transporters may play a decisive role in the co-evolution between plants and herbivores. Cardenolides, toxic steroid glycosides, are secondary plant metabolites that defend plants against herbivores by targeting their sodium–potassium ATPase. Despite their toxicity, several herbivorous insects such as the large milkweed bug (Oncopeltus fasciatus) have evolved adaptations to tolerate cardenolides and sequester them for their own defense. We investigate the role of two ABCB transporters of O. fasciatus for the paracellular transport of cardenolides by docking simulations and ATPase assays. Cardenolide binding of OfABCB1 and OfABCB2 is predicted by docking simulations and calculated binding energies are compared with substrate specificities determined in ATPase assays. Both tested ABCB transporters showed activity upon exposure to cardenolides and Km values that agreed well with the predictions of our docking simulations. We conclude that docking simulations can help identify transporter binding regions and predict substrate specificity, as well as provide deeper insights into the structural basis of ABC transporter function. Full article

Background/Objectives: Survival prospects following SARS-CoV-2 infection may extend beyond the acute phase, influenced by various factors including age, health conditions, and infection severity; however, this topic has not been studied in detail. Therefore, within this study, the mortality risk post-acute COVID-19 in the CRIT-COV-U cohort was investigated. Methods: Survival data from 651 patients that survived an acute phase of COVID-19 were retrieved and the association between urinary peptides and future death was assessed. Data spanning until December 2023 were collected from six countries, comparing mortality trends with age- and sex-matched COVID-19-negative controls. A death prediction classifier was developed and validated using pre-existing urinary peptidomic datasets. Results: Notably, 13.98% of post-COVID-19 patients succumbed during the follow-up, with mortality rates significantly higher than COVID-19-negative controls, particularly evident in younger individuals (<65 years). These data for the first time demonstrate that SARS-CoV-2 infection highly significantly increases the risk of mortality not only during the acute phase of the disease but also beyond for a period of about one year. In our study, we were further able to identify 201 urinary peptides linked to mortality. These peptides are fragments of albumin, alpha-2-HS-glycoprotein, apolipoprotein A-I, beta-2-microglobulin, CD99 antigen, various collagens, fibrinogen alpha, polymeric immunoglobulin receptor, sodium/potassium-transporting ATPase, and uromodulin and were integrated these into a predictive classifier (DP201). Higher DP201 scores, alongside age and BMI, significantly predicted death. Conclusions: The peptide-based classifier demonstrated significant predictive value for mortality in post-acute COVID-19 patients, highlighting the utility of urinary peptides in prognosticating post-acute COVID-19 mortality, offering insights for targeted interventions. By utilizing these defined biomarkers in the clinic, risk stratification, monitoring, and personalized interventions can be significantly improved. Our data also suggest that mortality should be considered as one possible symptom or a consequence of post-acute sequelae of SARS-CoV-2 infection, a fact that is currently overlooked. Full article

Sexually dimorphic traits such as growth and body size are often found in various crustaceans. Methyl farnesoate (MF), the main active form of sesquiterpenoid hormone in crustaceans, plays vital roles in the regulation of their molting and reproduction. However, understanding on the sex differences in their hormonal regulation is limited. Here, we carried out a comprehensive investigation on sexual dimorphic responses to MF in the hepatopancreas of the most dominant aquacultural crustacean—the white-leg shrimp (Litopenaeus vannamei). Through comparative transcriptomic analysis of the main MF target tissue (hepatopancreas) from both female and male L. vannamei, two sets of sex-specific and four sets of sex–dose-specific differentially expressed transcripts (DETs) were identified after different doses of MF injection. Functional analysis of DETs showed that the male-specific DETs were mainly related to sugar and lipid metabolism, of which multiple chitinases were significantly up-regulated. In contrast, the female-specific DETs were mainly related to miRNA processing and immune responses. Further co-expression network analysis revealed 8 sex-specific response modules and 55 key regulatory transcripts, of which several key transcripts of genes related to energy metabolism and immune responses were identified, such as arginine kinase, tropomyosin, elongation of very long chain fatty acids protein 6, thioredoxin reductase, cysteine dioxygenase, lysosomal acid lipase, estradiol 17-beta-dehydrogenase 8, and sodium/potassium-transporting ATPase subunit alpha. Altogether, our study demonstrates the sex differences in the hormonal regulatory networks of L. vannamei, providing new insights into the molecular basis of MF regulatory mechanisms and sex dimorphism in prawn aquaculture. Full article

Lithium is one of the lightest elements on Earth and it has been in the environment since the formation of the galaxy. While a common element, it has not been found to be an essential element in biological processes, ranging from single cell organisms to Homo sapiens. Instead, at an early stage of evolution, organisms committed to a range of elements such as sodium, potassium, calcium, magnesium, zinc, and iron to serve essential functions. Such ions serve critical functions in ion channels, as co-factors in enzymes, as a cofactor in oxygen transport, in DNA replication, as a storage molecule in bone and liver, and in a variety of other roles in biological processes. While seemingly excluded from a major essential role in such processes, lithium ions appear to be able to modulate a variety of biological processes and “correct” deviation from normal activity, as a deficiency of lithium can have biological consequences. Lithium salts are found in low levels in many foods and water supplies, but the effectiveness of Li salts to affect biological systems came to recent prominence with the work of Cade, who reported that administrating Li salts calmed guinea pigs and was subsequently effective at relatively high doses to “normalize” a subset of patients with bipolar disorders. Because of its ability to modulate many biological pathways and processes (e.g., cyclic AMP, GSK-3beta, inositol metabolism, NaK ATPases, neuro processes and centers, immune-related events, respectively) both in vitro and in vivo and during development and adult life, Li salts have become both a useful tool to better understand the molecular regulation of such processes and to also provide insights into altered biological processes in vivo during aging and in disease states. While the range of targets for lithium action supports its possible role as a modulator of biological dysregulation, it presents a conundrum for researchers attempting to elucidate its specific primary target in different tissues in vivo. This review will discuss aspects of the state of knowledge regarding some of the systems that can be influenced, focusing on those involving neural and autoimmunity as examples, some of the mechanisms involved, examples of how Li salts can be used to study model systems, as well as suggesting areas where the use of Li salts could lead to additional insights into both disease mechanisms and natural processes at the molecular and cell levels. In addition, caveats regarding lithium doses used, the strengths and weaknesses of rodent models, the background genetics of the strain of mice or rats employed, and the sex of the animals or the cells used, are discussed. Low-dose lithium may have excellent potential, alone or in combination with other interventions to prevent or alleviate aging-associated conditions and disease progression. Full article

The sodium pump, or Na⁺/K⁺-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na⁺) and potassium (K⁺) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA’s role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell–cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA β-subunits as cell adhesion molecules in glia and epithelial cells. Full article

Alzheimer’s disease (AD) is the most prevalent form of dementia among elderly people worldwide. Cerebrospinal fluid (CSF) is the optimal fluid source for AD biomarkers, while serum biomarkers are much more achievable. To search for novel diagnostic AD biomarkers, we performed a quantitative proteomic analysis of CSF and serum samples from AD and normal cognitive controls (NC). CSF and serum proteomes were analyzed via data-independent acquisition quantitative mass spectrometry. Our bioinformatic analysis was based on Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. In comparison to the controls, 8 proteins were more abundant in AD CSF, and 60 were less abundant in AD CSF, whereas 55 proteins were more and 10 were less abundant in the serum samples. ATPase-associated activity for CSF and mitochondrial functions for CSF and serum were the most enriched GO terms of the DEPs. KEGG enrichment analysis showed that the most significant pathways for the differentially expressed proteins were the N-glycan biosynthesis pathways. The area under the curve (AUC) values for CSF sodium-/potassium-transporting ATPase subunit beta-1 (AT1B1), serglycin (SRGN), and thioredoxin-dependent peroxide reductase, mitochondrial (PRDX3) were 0.867 (p = 0.004), 0.833 (p = 0.008), and 0.783 (p = 0.025), respectively. A panel of the above three CSF proteins accurately differentiated AD (AUC = 0.933, p = 0.001) from NC. The AUC values for serum probable phospholipid-transporting ATPase IM (AT8B4) and SRGN were moderate. The AUC of the CSF SRGN + serum SRGN was 0.842 (p = 0.007). These novel AD biomarker candidates are mainly associated with inflammation, ATPase activity, oxidative stress, and mitochondrial dysfunction. Further studies are needed to investigate the molecular mechanisms by which these potential biomarkers are involved in AD. Full article

High environmental hydrogen peroxide (H₂O₂) has been demonstrated to be toxic for fish. However, the response mechanism of fish to chronic H₂O₂ exposure is not yet well understood. Therefore, this study aimed to investigate the alteration in ion transport in gills and analyzed the potential response mechanism after chronic H₂O₂ exposure. The common carps were exposed to 0, 0.25, 0.50, and 1.00 mM of H₂O₂ for 14 days. The histopathological evaluation results indicated that H₂O₂ exposure caused incomplete gill filament structure. In the plasma, H₂O₂ exposure suppressed the potassium (K⁺) concentration but increased sodium (Na⁺) concentration. In the gills, the calcium (Ca²⁺) level was raised, but the K⁺ and chlorine (Cl⁻) levels were decreased after H₂O₂ exposure. After 14 days of exposure, H₂O₂ prompted the activities of Ca²⁺/Mg²⁺-ATPase and H⁺/K⁺-ATPase but suppressed Na⁺/K⁺-ATPase activity in the gills. Gene transcription analysis showed that the ion-regulation-related genes including nkaa and rhbg were downregulated after H₂O₂ exposure. In addition, H₂O₂ exposure upregulated the mRNA levels of cam and camk II, indicating that the Ca²⁺ singling pathway was activated. In conclusion, our data showed that chronic H₂O₂ exposure altered gill structure and disturbed ion transport, which further negatively affected the equilibrium of ions and osmotic pressure. Full article

The Na⁺/K⁺ ATPase (NKA) is present in the cellular membrane of most eukaryotic cells. It utilizes energy released by ATP hydrolysis to pump sodium ions out of the cell and potassium ions into the cell, which establishes and controls ion gradients. Functional NKA pumps consist of three subunits, alpha, beta, and FXYD. The alpha subunit serves as the catalytic subunit while the beta and FXYD subunits regulate the proper folding and localization, and ion affinity of the alpha subunit, respectively. Here we demonstrate that knockdown of NKA beta subunit 2 mRNA (nkaβ2) reduces fecundity in female Ae. aegypti. We determined the expression pattern of nkaβ2 in several adult mosquito organs using qRT-PCR. We performed RNAi-mediated knockdown of nkaβ2 and assayed for lethality, and effects on female fecundity. Tissue expression levels of nkaβ2 mRNA were highest in the ovaries with the fat body, midgut and thorax having similar expression levels, while Malpighian tubules had significantly lower expression. Survival curves recorded post dsRNA injection showed a non-significant decrease in survival of nkaβ2 dsRNA-injected mosquitoes compared to GFP dsRNA-injected mosquitoes. We observed a significant reduction in the number of eggs laid by nkaβ2 dsRNA-injected mosquitoes compared to control mosquitoes. These results, coupled with the tissue expression profile of nkaβ2, indicate that this subunit plays a role in normal female Ae. aegypti fecundity. Additional research needs to be conducted to determine the exact role played by NKAβ2 in mosquito post-blood meal nutrient sensing, transport, yolk precursor protein (YPP) synthesis and yolk deposition. Full article

Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers. Full article

EGF plays an important role in the intestinal repair and nutrients transport of animals. However, the effect of EGF on the intestinal health of piglets with IUGR has not been reported. Thus, the present study was performed to investigate the effects of EGF on the intestinal morphology, glucose absorption, antioxidant capacity, and barrier function of piglets with IUGR. A total of 6 NBW piglets and 12 IUGR piglets were randomly divided into three treatments: NC group (NBW piglets fed with basal diet, n = 6), IC group (IUGR piglets fed with basal diet, n = 6), and IE group (IUGR piglets fed with basal diet supplemented with 2 mg/kg EGF, n = 6). Growth performance, serum biochemical profile, jejunum histomorphology, jejunum glucose absorption and antioxidant capacity, and jejunal barrier function were measured. The results showed that EGF supplementation significantly increased the final body weight (FBW), average daily gain (ADG), and average daily feed intake (ADFI) of piglets with IUGR; EGF supplementation significantly increased the total protein (TP), glucose (GLU), and immunoglobulin G (IgG) levels compared with the IUGR piglets in the IC group; EGF administration effectively exhibited an increased jejunum villus height (VH) and the villus-height-to-crypt-depth ratio (V/C) of IUGR piglets compared with the IC group; EGF supplementation significantly increased sodium/potassium-transporting adenosine triphosphatase (Na⁺/K⁺-ATPase) activity, intestinal alkaline phosphatase (AKP) activity, glucose transporter sodium/glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), and AMP-activated protein kinase α1 (AMPK-α1) mRNA expressions in the jejunum of IUGR piglets compared with the IC group; EGF supplementation exhibited increased superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) levels, tended to increase glutathione peroxidase (GSH-Px) and catalase (CAT) activities, and tended to decrease the malondialdehyde (MDA) level in the jejunum of IUGR piglets compared with the IC group; EGF supplementation significantly increased ZO-1, Claudin-1, Occludin, and MUC2 mRNA expressions and improved secreted immunoglobulin A (sIgA) secretion in the jejunum of IUGR piglets compared with the IC group and tended to decrease the interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) levels in the jejunum of IUGR piglets compared with the IC group. Pearson’s correlation analysis further showed that EGF can promote intestinal development and nutrient absorption by promoting intestinal barrier function, thus improving the growth performance of IUGR piglets. Full article

Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage. Full article