The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis.

In brief: Although common in many commercial extenders, supraphysiological concentrations of glucose in the media may be detrimental to stallion spermatozoa. In this study, we present evidence that these elevated glucose levels may predispose spermatozoa to ferroptosis. Abstract: Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP; however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists. The composition of the media in which the spermatozoa are extended, or exposed to in the mare's reproductive tract, exerts a profound effect on sperm function and may even accelerate cell demise. Recent research indicates that high concentrations of glucose in the media, although common in commercial extenders, may be detrimental. To determine if supraphysiological glucose concentration may induce or predispose to ferroptosis (a caspase-independent form of programmed cell death, triggered by oxidative stress), stallion spermatozoa were incubated under different concentrations of glucose, 67 mM (HG) or 1 mM plus 10 mM pyruvate (LG-HP), in the presence or absence of known inductors of ferroptosis. Furthermore, we developed a single-cell flow metabolic assay to identify different metabolic phenotypes in spermatozoa. Storage and incubation of spermatozoa under high glucose concentrations led to an increase in the percentage of necrotic spermatozoa (P < 0.0001). Moreover, ferroptosis was induced more intensely in sperm in media with high glucose concentrations (P < 0.0001). Finally, we observed that induction of ferroptosis modified two proteins (oxoglutarate dehydrogenase and superoxide dismutase 2) in spermatozoa incubated in media containing 67 mM glucose but not in media containing 1 mM glucose and 10 mM pyruvate. The composition of the media, especially the concentration of glucose, exerts a major impact on the functionality and life span of the spermatozoa. The results reported here may pave the way for improvements in existing semen extenders.

Phosphoproteomics for the identification of new mechanisms of cryodamage: the role of SPATA18 in the control of stallion sperm function†.

Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protein 4 (AKAP4), A-kinase anchoring protein 3 (AKAP3) and the Family with sequence similarity 71 member B (FAM71B). These proteins belong to the gene ontology (GO) terms sperm fibrous sheath (GO: 0035686), and sperm principal piece (GO: 0097228). The regulatory interactions between kinases and phosphorylation sites on the proteins that were affected most were also investigated, and the potential kinases (based on human orthologs) involved in the regulation of these phosphoproteins identified were: PKCß for SPATA18 and GSK3ß for CABYR. Kinase inhibition assays were also conducted showing that kinases phosphorylating the above-mentioned proteins play an important role in their activity and thus, phosphorylation controls the activity of these proteins and their role in the regulation of the functionality and viability of stallion spermatozoa. In conclusion, the data reported here contribute to the understanding of the fact that the dephosphorylation of certain proteins is a molecular lesion induced by cryopreservation in the stallion spermatozoa.

Advances in the ultrasound diagnosis in equine reproductive medicine: New approaches.

Ultrasound technology has led to new lines of research in equine reproduction, and it has helped to greatly improve clinical diagnosis and reproductive outcomes in equine practice. This review aims to discuss the potential clinical uses and new approaches of ultrasonography in equine reproduction. Doppler modalities are usually used to evaluate the vascularization of the follicles, corpus luteum (CL), and the uterus in the mare for diagnostic purposes. Inclusion of Doppler ultrasound in artificial insemination and embryo transfer programs could improve the reproductive outcome of these techniques. Better selection of recipients based on CL functionality, early pregnancy diagnosis 7-8 days postovulation of the donor before flushing or diagnosis of mares with endometritis with pathological increases of blood flow are examples of clinical applications in the mare. In the stallion, colour Doppler ultrasound has improved the diagnostic potential of B-mode ultrasound, improving the differential diagnosis of pathologies such as testicular torsion (decrease or absence of blood flow in the cord) and orchitis (increased blood flow in the cord). The incorporation of pulsed Doppler ultrasound into the reproductive evaluation of the stallion has enabled early identification of stallions with testicular dysfunction, thus allowing administration of timely treatment and subsequent improvements of the fertility prognosis for these animals. In addition, this technique has been used in the monitoring of patients undergoing medical and surgical treatments, thus verifying their efficacy. Recently, computer-assisted pixel analysis using specific software has been performed in research work in order to semi-quantitatively evaluate the vascularization (colour and power Doppler) and echotexture of different organs. These softwares are now being developed for clinical purposes, as is the case with Ecotext, a computer program developed for the evaluation of testicular echotexture, providing information on testicular functionality.

In Stallion Spermatozoa, Superoxide Dismutase (Cu-Zn) (SOD1) and the Aldo-Keto-Reductase Family 1 Member b (AKR1B1) Are the Proteins Most Significantly Reduced by Cryopreservation.

Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a split sample design, 30 ejaculates from 10 different stallions were analyzed as fresh spermatozoa, and another aliquot from the same ejaculate was analyzed as a frozen thawed sample. The proteome was studied under both conditions using UHPLC-MS/MS and bioinformatic analysis conducted to identify discriminant variables between both conditions. Data are available through the ProteomeXchange Consortium with identifier PXD022236. The proteins most significantly reduced were Aldo-keto reductase family 1 member B (p = 2.2 × 10-17) and Superoxide dismutase (Cu-Zn) (p = 4.7 × 10-14). This is the first time that SOD1 has been identified as a discriminating variable using bioinformatic analysis, where it was one of the most highly significantly different proteins seen between fresh and frozen thawed semen. This finding strongly supports the theory that alteration in redox regulation and oxidative stress is a major factor involved in cryodamage and suggests that control of redox regulation should be a major target to improve current cryopreservation procedures.

Differences in the proteome of stallion spermatozoa explain stallion-to-stallion variability in sperm quality post-thaw†.

The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh aliquot and the other half was frozen and then analyzed as a frozen-thawed aliquot. Computer-assisted sperm analysis and flow cytometry were used to analyze sperm quality. Detailed proteomic analysis was performed on fresh and frozen and thawed aliquots, and bioinformatic analysis was used to identify discriminant variables in fresh samples able to predict the outcome of cryopreservation. Those with a fold change > 3, a P = 8.2e-04, and a q = 0.074 (equivalent to False discovery rate (FDR)) were selected, and the following proteins were identified in fresh samples as discriminant variables of good motility post-thaw: F6YTG8, K9K273, A0A3Q2I7V9, F7CE45, F6YU15, and F6SKR3. Other discriminant variables were also identified as predictors of good mitochondrial membrane potential and viability post-thaw. We concluded that proteomic approaches are a powerful tool to improve current sperm biotechnologies.

Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation, and stallion sperm function†.

Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain in the mitochondria appears to be crucial for both sperm motility and maintaining viability, whereas glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that Gene Ontology terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were predominant. Following this, the hypothesis that low glucose concentrations can provide sufficient support for motility and velocity, and thus glucose concentration can be significantly reduced in the medium, was tested. Aliquots of stallion semen in four different media were stored for 48 h at 18°C; a commercial extender containing 67 mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1 mM) and high pyruvate (10 mM) (LG-HP) sustained better motility and velocities than those stored in the commercial extender formulated with very high glucose (61.7 ± 1.2% in INRA 96 vs 76.2 ± 1.0% in LG-HP media after 48 h of incubation at 18°C; P < 0.0001). Moreover, mitochondrial activity was superior in LG-HP extenders (24.1 ± 1.8% in INRA 96 vs 51.1 ± 0.7% in LG-HP of spermatozoa with active mitochondria after 48 h of storage at 18°C; P < 0.0001). Low glucose concentrations may permit more efficient sperm metabolism and redox regulation when substrates for an efficient tricarboxylic acid cycle are provided. The improvement seen using low glucose extenders is due to reductions in the levels of glyoxal and methylglyoxal, 2-oxoaldehydes formed during glycolysis; these compounds are potent electrophiles able to react with proteins, lipids, and DNA, causing sperm damage.

Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration†.

Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI.

Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa.

The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R2 = 0.38, P ≤ 0.01; HK1 R2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001; HK1 R2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R2 = 0.29, P ≤ 0.05; HK1 R2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R2 = 0.50, P ≤ 0.01) and straightness (HK1 R2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.

Age-Induced Differential Changes in the Central and Colonic Human Circadian Oscillators.

Aging modifies not only multiple cellular and homeostatic systems, but also biological rhythms. The circadian system is driven by a central hypothalamic oscillator which entrains peripheral oscillators, in both cases underlain by circadian genes. Our aim was to characterize the effect of aging in the circadian expression of clock genes in the human colon. Ambulatory recordings of the circadian rhythms of skin wrist temperature, motor activity and the integrated variable TAP (temperature, activity and position) were dampened by aging, especially beyond 74 years of age. On the contrary, quantitative analysis of genes expression in the muscle layer of colonic explants during 24 h revealed that the circadian expression of Bmal1, Per1 and Clock genes, was larger beyond that age. In vitro experiments showed that aging induced a parallel increase in the myogenic contractility of the circular colonic muscle. This effect was not accompanied by enhancement of Ca2+ signals. In conclusion, we describe here for the first time the presence of a molecular oscillator in the human colon. Aging has a differential effect on the systemic circadian rhythms, that are impaired by aging, and the colonic oscillator, that is strengthened in parallel with the myogenic contractility.

The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoa†.

Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P < 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation-reduction potential of spermatozoa, with sulfasalazine only preventing this effect on fresh spermatozoa that were incubated for 3 h at 37°C, but not in frozen-thawed spermatozoa. While sulfasalazine reduced the motility of frozen-thawed spermatozoa, it increased motility in fresh samples. The present findings provide new and relevant data on the mechanism regulating the redox status of spermatozoa and suggest that a different redox regulatory mechanism exists in cryopreserved spermatozoa, thus providing new clues to improve current cryopreservation technologies and treat male factor infertility.

Characterization of the motor inhibitory role of colonic mucosa under chemical stimulation in mice.

The main roles of the colonic mucosa are the absorption of water and electrolytes and the barrier function that preserves the integrity of the colonic wall. The mediators and mechanisms to accomplish these functions are under continuous investigation, but little attention has been paid to a possible control of colonic motility by the mucosa that would fine tune the relationship between absorption and motility. The purpose of this study was to establish the role of the mucosa in the control of induced colonic contractility. Young ICR-CD1 mice (3-5 mo old) were studied. Isometric tension transducers were used to record contractility in full-thickness (FT) and mucosa-free (MF) strips from proximal colon. Proximal FT strips showed lower KCl- and bethanechol-induced responses than MF strips. The difference was not due to mechanical artefacts since the contractile response of FT strips to electrical field stimulation was around 50% lower than in MF. The inhibitory effects of the mucosa on FT strips were mimicked by immersion of separate strips of mucosa in the organ bath but not by addition of mucosal extract, suggesting gaseous molecules as mediators of this effect. Incubation of MF strips with synthase inhibitors of nitric oxide, carbon monoxide, and hydrogen sulfide abolished the inhibition caused by addition of the mucosal strip, indicating that mucosal gasotransmitters are the mediators of these effects. This suggests that the control of colonic motility exerted by the mucosa could fine tune the balance between transit and absorption.

Age-related changes in mitochondrial function of mouse colonic smooth muscle: beneficial effects of melatonin.

Aging is a multifactorial process that involves biochemical, structural, and functional changes in mitochondria. The ability of melatonin to palliate the alterations induced by aging is based on its chronobiologic, antioxidant, and mitochondrial effects. There is little information about the effects of melatonin on the in situ mitochondrial network of aging cells and its physiological implications. We have studied the ability of melatonin to prevent the functional alterations of in situ mitochondria of smooth muscle cells and its impact on contractility. Mitochondrial membrane potential was recorded in isolated colonic smooth muscle cells from young mice (3 month old), aged mice (22-24-month old), and aged mice treated with melatonin (starting at 14-month age). Aging induced a partial mitochondrial depolarization in resting conditions and reduced the depolarizing response to cellular stimulation. Use of oligomycin indicated that aging enhanced the resting activity of the mitochondrial ATP synthase, whereas in young cells, the enzyme operated mainly in reverse mode. Melatonin treatment prevented all these changes. Aging reduced both spontaneous and stimulated contraction of colonic strips and shifted the metabolic dependence of contraction from mitochondria to glycolysis, as indicated the use of mitochondrial and glycolysis inhibitors. These functional alterations were also palliated by melatonin treatment. Aging effects were not related to a decrease in Ca2+ store mobilization, because this was enhanced in aged cells and restored by melatonin. In conclusion, melatonin prevents the age induced in situ mitochondrial potential alterations in smooth muscle cells and the associated changes in contractility and metabolism.

Pyruvate enhances stallion sperm function in high glucose media improving overall metabolic efficiency.

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile.

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

Proteomic research on new urinary biomarkers of renal disease in canine leishmaniosis: Survival and monitoring response to treatment.

The objective of our study was to search for survival biomarkers (SB) and treatment response monitoring biomarkers (TRMB) in the urinary proteome of dogs with renal disease secondary to canine leishmaniosis (CanL), using UHPLC-MS/MS. The proteomic data are available via ProteomeXchange with identifier PXD042578. Initially, a group of 12 dogs was evaluated and divided into survivors (SG; n = 6) and nonsurvivors (NSG; n = 6). A total of 972 proteins were obtained from the evaluated samples. Then, bioinformatic analysis reduced them to 6 proteins like potential SB increased in the NSG, specifically, Haemoglobin subunit Alpha 1, Complement Factor I, Complement C5, Fibrinogen beta chain (fragment), Peptidase S1 domain-containing protein, and Fibrinogen gamma chain. Afterwards, SG was used to search for TRMB, studying their urine at 0, 30, and 90 days, and 9 proteins that decreased after treatment were obtained: Apolipoprotein E, Cathepsin B, Cystatin B, Cystatin-C-like, Lysozyme, Monocyte differentiation CD14, Pancreatitis-associated precursor protein, Profilin, and Protein FAM3C. Finally, enrichment analysis provided information about the biological mechanisms in which these proteins are involved. In conclusion, this study provides 15 new candidate urinary biomarkers and an improved understanding of the pathogenesis of kidney disease in CanL.

An integrated overview on the regulation of sperm metabolism (glycolysis-Krebs cycle-oxidative phosphorylation).

An overview of the sperm metabolism is presented; using the stallion as a model we review glycolysis, Krebs Cycle and oxidative phosphorylation, paying special attention to the interactions among them. In addition, metabolism implies a series of coordinated oxidation-reduction reactions and in the course of these reactions reactive oxygen species (ROS) and reactive oxoaldehydes are produced ; the electron transport chain (ETC) in the mitochondria is the main source of the anion superoxide and hydrogen peroxide, while glycolysis produces 2-oxoaldehydes such as methylglyoxal as byproducts; due to the adjacent carbonyl groups are strong electrophiles (steal electrons oxidizing other compounds). Sophisticated mechanisms exist to maintain redox homeostasis, because ROS under controlled production also have important regulatory functions in the spermatozoa. The interactions between metabolism and production of reactive oxygen species are essential for proper sperm function, and deregulation of these processes rapidly leads to sperm malfunction and finally death. Lastly, we briefly describe two techniques that will expand our knowledge on sperm metabolism in the coming decades, metabolic flow cytometry and the use of the "omics" technologies, proteomics and metabolomics, specifically the micro and nano proteomics/metabolomics. A better understanding of the metabolism of the spermatozoa will lead to big improvements in sperm technologies and the diagnosis and treatment of male factor infertility.

Urinary proteome of dogs with renal disease secondary to leishmaniosis.

Canine leishmaniosis is frequently associated with the development of renal disease. Its pathogenesis is complex and not fully understood. For this reason, this study aimed to describe the urinary proteome, and identify possible new biomarkers in dogs with kidney disease secondary to leishmaniosis. Urine samples were collected from 20 dogs, 5 from healthy dogs, and 15 from stages Leishvet III and IV. Urine samples were analyzed by UHPLC-MS/MS. The data are available via ProteomeXchange with identifier PXD029165. A total of 951 proteins were obtained. After bioinformatic analysis, 93 urinary proteins were altered in the study group. Enrichment analysis performed on these proteins showed an overrepresentation of the complement activation pathway, among others. Finally, 12 discriminant variables were found in dogs with renal disease secondary to leishmaniosis, highlighting C4a anaphylatoxin, apolipoprotein A-I, haptoglobin, leucine-rich alpha-2-glycoprotein 1, and beta-2-microglobulin. This study is the first to describe the urinary proteomics of dogs with renal disease caused by leishmaniosis, and it provides new possible biomarkers for the diagnosis and monitoring of this disease.

The seminal plasma proteins Peptidyl arginine deaminase 2, rRNA adenine N (6)-methyltransferase and KIAA0825 are linked to better motility post thaw in stallions.

Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our laboratory. Aliquots of the ejaculate were separated and stored at -80 °C until further proteomic analysis. Semen analysis was performed using computer assisted sperm analysis and flow cytometry. Significant differences in proteome composition of seminal plasma were observed in the group of stallions showing better motility post thaw. 3116 proteins were identified, and of these, 34 were differentially expressed in stallions with better motility post thaw, 4 of them were also differentially expressed in stallions with different percentages of linearly motile sperm post thaw and 1 protein, Midasin, was expressed in stallions showing high circular velocity post thaw. Seminal plasma proteins may play a major role in sperm functionality; being vehiculated through extracellular vesicles and participating in sperm physiology. Bioinformatic analysis identifies discriminant proteins able to predict the outcome of cryopreservation, identifying potential new biomarkers to assess ejaculate quality.

Seminal plasma proteins as potential biomarkers for sperm motility and velocities.

Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm analysis (CASA), while protein composition of the seminal plasma was studied using UHPLC-MS/MS. Specific proteins were more abundant in samples with poorer percentages of total motility, average path velocity and circular velocity, and were: Secreted phosphoprotein 1, Fructose-bisphosphate aldolase (p = 1,95E-09; q = 0.0005) and Malate dehydrogenase 1 (p = 1,41E-11; q = 0.002), to the contrary samples with better straight-line velocity values were enriched in Glutathione peroxidase (p=0.00013; q=0.04) and Triosephosphate isomerase (p=0.00015; q=0.04).

Dataset of the sperm proteome of stallions with different motility.

This paper provides a detailed set of data on how the stallion sperm proteome differs among stallions with different sperm motilities, although within normal ranges. Findings distinguish proteins that may help to identify stallions of superior sperm motility. Sperm proteins were analyzed using a UHPLC/MS/MS system comprising of an Agilent 1290 infinity series UHPLC coupled to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). These data can be used to disclose potential targets to identify good sperm samples and to study specific pathways involved in the regulation of sperm motility. This data article is linked to the paper "Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility Journal of Proteomics 247:104335 doi: 10.1016/j.jprot.2021.104335".