Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis.

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8-iso-PGF2α and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.

Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes.

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm.

BACKGROUND: Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. RESULTS: The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. CONCLUSIONS: We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the use of Androcoll-Bear 65% since the yield is better than Androcoll-Bear 80%. Our findings pave the way for further research on application of sperm selection techniques to sperm banking in the brown bear.

Flow cytometry in Spermatology: A bright future ahead.

Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high-throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information.The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single-cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry-based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.

Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation.

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na+/K+ gradient, which is dependent on an Na+-K+ antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA.

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre- and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.

Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-ß-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 µm) and EGCG (10, 20 and 60 µm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 µm (but not of 60 µm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 µm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.

The Impact of Reproductive Technologies on Stallion Mitochondrial Function.

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen-thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.

Molecular mechanisms of quinolone, macrolide, and tetracycline resistance among Campylobacter isolates from initial stages of broiler production.

The aim of this study was to investigate the resistance mechanisms of quinolones, macrolides and tetracycline in campylobacter isolates from grandparent and parent broiler breeders in Spain. Twenty-six isolates were investigated for quinolone resistance, three isolates for macrolide resistance and 39 for tetracycline resistance. All of the quinolone-resistant isolates possessed the mutation Thr86Ile in the quinolone resistance-determining region of gyrA and one isolate possessed the mutation Pro104Ser. Only one Campylobacter coli population (defined by restriction fragment length polymorphism-polymerase chain reaction of flaA and pulsed field gel electrophoresis) was resistant to erythromycin, and the mutation A2075G (23S rDNA) was responsible for macrolide resistance. The tetO gene was found in all of the tetracycline-resistant isolates. Twenty-two out of the 39 isolates investigated by Southern blot possessed chromosomic location of tetO and 17 were located on plasmids. Most of the plasmids with tetO were of around 60 kb and conjugation was demonstrated in a selection of them. In conclusion, we showed that Thr86Ile is highly prevalent in quinolone-resistant isolates as well as mutation A2075G in macrolide-resistant isolates of poultry origin. More variability was found for tetO. The possibility of horizontal transmission of tetO among campylobacter isolates is also an issue of concern in public health.

Use of colour and spectral Doppler ultrasonography in stallion andrology.

Doppler ultrasonography is an important tool in the andrological evaluation in humans; however, this method is not so extensively used by equine andrologists. Spectral or pulse Doppler is a useful non-invasive method for the early diagnosis of subfertility problems in the male, especially those triggered by vascular disturbance. The identification of any disturbance in the blood flow of the testis is crucial for a correct diagnosis of various testicular and scrotal disorders but also to monitor the therapeutic outcome following treatment. The aim of this review is to provide an update on the current use of colour and spectral Doppler ultrasound in stallion andrology, and to promote the use of this technique during the soundness reproductive examination of the stallion, as this particular branch of reproductive medicine is receiving increasing interest.

Standing laparoscopic peritoneal flap hernioplasty of the vaginal rings does not modify the sperm production and motility characteristics in intact male horses.

Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this procedure based on 1-year follow-ups. There were no significant differences in the measured sperm variables (assessments based on the DSO, MOT, PMOT, VSL, VCL and VAP) during 1-year follow-ups.

Effect of overnight staining on the quality of flow cytometric sorted stallion sperm: comparison with tradtitional protocols.

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.

Caspase activation, hydrogen peroxide production and Akt dephosphorylation occur during stallion sperm senescence.

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.

Identification of apoptotic bodies in equine semen.

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37 °C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin-V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer-assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.

Bacterial endometritis-induced changes in the endometrial proteome in mares: Potential uterine biomarker for bacterial endometritis.

Equine endometritis is one of the main causes of subfertility in the mare. Unraveling the molecular mechanisms involved in this condition and pinpointing proteins with biomarker potential could be crucial in both diagnosing and treating this condition. This study aimed to identify the endometritis-induced changes in the endometrial proteome in mares and to elucidate potential biological processes in which these proteins may be involved. Secondly, biomarkers related to bacterial endometritis (BE) in mares were identified. Uterine lavage fluid samples were collected from 28 mares (14 healthy: negative cytology and culture, and no clinical signs and 14 mares with endometritis: positive cytology and culture, in addition to clinical signs). Proteomic analysis was performed with a UHPLC-MS/MS system and bioinformatic analysis was carried out using Qlucore Omics Explorer. Gene Ontology enrichment and pathway analysis (PANTHER and KEGG) of the uterine proteome were performed to identify active biological pathways in enriched proteins from each group. Quantitative analysis revealed 38 proteins differentially abundant in endometritis mares when compared to healthy mares (fold changes >4.25, and q-value = 0.002). The proteins upregulated in the secretome of mares with BE were involved in biological processes related to the generation of energy and REDOX regulation and to the defense response to bacterium. A total of 24 biomarkers for BE were identified using the biomarker workbench algorithm. Some of the proteins identified were related to the innate immune system such as isoforms of histones H2A and H2B involvement in neutrophil extracellular trap (NET) formation, complement C3a, or gelsolin and profilin, two actin-binding proteins which are essential for dynamic remodeling of the actin cytoskeleton during cell migration. The other group of biomarkers were three known antimicrobial peptides (lysosome, equine cathelicidin 2 and myeloperoxidase (MPO)) and two uncharacterized proteins with a high homology with cathelicidin families. Findings in this study provide the first evidence that innate immune cells in the equine endometrium undergo reprogramming of metabolic pathways similar to the Warburg effect during activation. In addition, biomarkers of BE in uterine fluid of mares including the new proteins identified, as well as other antimicrobial peptides already known, offer future lines of research for alternative treatments to antibiotics.

Consequences of butylated hydroxytoluene in the freezing extender on post-thaw characteristics of stallion spermatozoa in vitro.

Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant effect on any of the sperm parameters evaluated post-thaw.

The membrane of the mammalian spermatozoa: much more than an inert envelope.

Sperm plasma membrane is a very important structure that functions to protect sperm against extracellular injuries and to respond to physiological challenges. It plays a crucial role during sperm capacitation, in sperm-egg interaction and, finally, in fertilization. Concerning sperm technology, possibly the most important factors causing damage in mammalian spermatozoa membranes are initiated by the osmotic stress generated by dehydration of the cells during freezing and thawing. These changes are rapidly derived to the plasma and organelle membranes that gradually experiment loss of membrane architecture, causing unbalanced production of reactive oxygen species and increased lipid peroxidation. Other procedures such as sperm sorting or liquid storage of sperm also induce harmful changes in the integrity of the membrane. The specific composition of lipids of the sperm membranes may provide clues for understanding the mechanisms behind the differences found in the response to stress in different species. In the present review, we deal with the composition, architecture and organization of the sperm plasma membrane, emphasizing the factors that can affect membrane integrity. The intracellular signalling pathways related with membrane reorganization during capacitation and acrosome reaction are also reviewed.

Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage.

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer-assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro-1) and mitochondrial membrane potential (JC-1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL-2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL-2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL-2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro-1 negative) sperm post-thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.

Protein oxidation marker, α-amino adipic acid, impairs proteome of differentiated human enterocytes: Underlying toxicological mechanisms.

Protein oxidation and oxidative stress are involved in a variety of health disorders such as colorectal adenomas, inflammatory bowel's disease, neurological disorders and aging, among others. In particular, the specific final oxidation product from lysine, the α-amino adipic acid (α-AA), has been found in processed meat products and emphasized as a reliable marker of type II diabetes and obesity. Currently, the underlying mechanisms of the biological impairments caused by α-AA are unknown. To elucidate the molecular basis of the toxicological effect of α-AA, differentiated human enterocytes were exposed to dietary concentrations of α-AA (200 µM) and analyzed by flow cytometry, protein oxidation and proteomics using a Nanoliquid Chromatography-Orbitrap MS/MS. Cell viability was significantly affected by α-AA (p < 0.05). The proteomic study revealed that α-AA was able to alter cell homeostasis through impairment of the Na+/K+-ATPase pump, energetic metabolism, and antioxidant response, among other biological processes. These results show the importance of dietary oxidized amino acids in intestinal cell physiology and open the door to further studies to reveal the impact of protein oxidation products in pathological conditions.