Proteomics is advancing the understanding of stallion sperm biology.

The mammalian ejaculate is very well suited to proteomics studies. As such, research concerning sperm proteomics is offering a huge amount of new information on the biology of spermatozoa. Among domestic animals, horses represent a species of special interest, in which reproductive technologies and a sizeable market of genetic material have grown exponentially in the last decade. Studies using proteomic approaches have been conducted in recent years, showing that proteomics is a potent tool to dig into the biology of the stallion spermatozoa. The aim of this review is to present an overview of the research conducted, and how these studies have improved our knowledge of stallion sperm biology. The main outcomes of the research conducted so far have been an improved knowledge of metabolism, and its importance in sperm functions, the impact of different technologies on the sperm proteome, and the identification of potential biomarkers. Moreover, proteomics of seminal plasma and phosphoproteomics are identified as areas of major interest.

Midday water use efficiency in sorghum is linked to faster stomatal closure rate, lower stomatal aperture and higher stomatal density.

Most studies assume midday gas exchange measurements capture the leaf's daytime performance. However, stomatal conductance (gs ) and photosynthesis (An ) fluctuate diurnally due to endogenous and environmental rhythms, which can affect intrinsic water use efficiency (iWUE). Six Sorghum lines with contrasting stomatal anatomical traits were grown in environmentally controlled conditions, and leaf gas exchange was measured three times a day. Stomatal anatomy and kinetic responses to light transients were also measured. The highest An and gs and the lowest iWUE were observed at midday for most lines. Diurnally averaged iWUE correlated positively with morning and midday iWUE and negatively with the time taken for stomata to close after transition to low light intensity (kclose ). There was significant variation among sorghum lines for kclose , and smaller kclose correlated with lower gs and higher stomatal density (SD) across the lines. In turn, gs was negatively correlated with SD and regulated by the operational stomatal aperture regardless of stomatal size. Altogether, our data suggest a common physiology to improve iWUE in sorghum related to the control of water loss without impacting photosynthesis relying on higher SD, lower stomatal aperture and faster stomatal closing in response to low light intensity.

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.

Real-world evidence on the dosing and safety of C.E.R.A. in pediatric dialysis patients: findings from the International Pediatric Dialysis Network registries.

BACKGROUND: This retrospective real-world study used data from two registries, International Pediatric Peritoneal Dialysis Network (IPPN) and International Pediatric Hemodialysis Network (IPHN), to characterize the efficacy and safety of continuous erythropoietin receptor activator (C.E.R.A.) in pediatric patients with chronic kidney disease (CKD) on peritoneal dialysis (PD) or hemodialysis (HD). METHODS: IPPN and IPHN collect prospective data (baseline and every 6 months) from pediatric PD and HD centers worldwide. Demographics, clinical characteristics, dialysis information, treatment, laboratory parameters, number and causes of hospitalization events, and deaths were extracted for patients on C.E.R.A. treatment (IPPN: 2007-2021; IPHN: 2013-2021). RESULTS: We analyzed 177 patients on PD (median age 10.6 years) and 52 patients on HD (median age 14.1 years) who had ≥ 1 observation while being treated with C.E.R.A. The median (interquartile range [IQR]) observation time under C.E.R.A. exposure was 6 (0-12.5) and 12 (0-18) months, respectively. Hemoglobin concentrations were stable over time; respective means (standard deviation) at last observation were 10.9 (1.7) g/dL and 10.4 (1.7) g/dL. Respective median (IQR) monthly C.E.R.A. doses at last observation were 3.5 (2.3-5.1) µg/kg, or 95 (62-145) µg/m2 and 2.1 (1.2-3.4) µg/kg, or 63 (40-98) µg/m2. Non-elective hospitalizations occurred in 102 (58%) PD and 32 (62%) HD patients. Seven deaths occurred (19.8 deaths per 1000 observation years). CONCLUSIONS: C.E.R.A. was associated with efficient maintenance of hemoglobin concentrations in pediatric patients with CKD on dialysis, and appeared to have a favorable safety profile. The current analysis revealed no safety signals.

Microbiota-Derived Extracellular Vesicles as a Postbiotic Strategy to Alleviate Diarrhea and Enhance Immunity in Rotavirus-Infected Neonatal Rats.

Rotavirus (RV) infection is a major cause of acute gastroenteritis in children under 5 years old, resulting in elevated mortality rates in low-income countries. The efficacy of anti-RV vaccines is limited in underdeveloped countries, emphasizing the need for novel strategies to boost immunity and alleviate RV-induced diarrhea. This study explores the effectiveness of interventions involving extracellular vesicles (EVs) from probiotic and commensal E. coli in mitigating diarrhea and enhancing immunity in a preclinical model of RV infection in suckling rats. On days 8 and 16 of life, variables related to humoral and cellular immunity and intestinal function/architecture were assessed. Both interventions enhanced humoral (serum immunoglobulins) and cellular (splenic natural killer (NK), cytotoxic T (Tc) and positive T-cell receptor γδ (TCRγδ) cells) immunity against viral infections and downregulated the intestinal serotonin receptor-3 (HTR3). However, certain effects were strain-specific. EcoR12 EVs activated intestinal CD68, TLR2 and IL-12 expression, whereas EcN EVs improved intestinal maturation, barrier properties (goblet cell numbers/mucin 2 expression) and absorptive function (villus length). In conclusion, interventions involving probiotic/microbiota EVs may serve as a safe postbiotic strategy to improve clinical symptoms and immune responses during RV infection in the neonatal period. Furthermore, they could be used as adjuvants to enhance the immunogenicity and efficacy of anti-RV vaccines.

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.

Anatomical drivers of stomatal conductance in sorghum lines with different leaf widths grown under different temperatures.

Sustaining crop productivity and resilience in water-limited environments and under rising temperatures are matters of concern worldwide. We investigated the leaf anatomical traits that underpin our recently identified link between leaf width (LW) and intrinsic water use efficiency (iWUE), as traits of interest in plant breeding. Ten sorghum lines with varying LW were grown under three temperatures to expand the range of variation of both LW and gas exchange rates. Leaf gas exchange, surface morphology and cross-sectional anatomy were measured and analysed using structural equations modelling. Narrower leaves had lower stomatal conductance (gs ) and higher iWUE across growth temperatures. They also had smaller intercellular airspaces, stomatal size, percentage of open stomatal aperture relative to maximum, hydraulic pathway, mesophyll thickness, and leaf mass per area. Structural modelling revealed a developmental association among leaf anatomical traits that underpinned gs variation in sorghum. Growing temperature and LW both impacted leaf gas exchange rates, but only LW directly impacted leaf anatomy. Wider leaves may be more productive under well-watered conditions, but consume more water for growth and development, which is detrimental under water stress.

Elevated [CO2] negatively impacts C4 photosynthesis under heat and water stress without penalizing biomass.

Elevated [CO2] (eCO2) and water stress reduce leaf stomatal conductance (gs), which may affect leaf thermoregulation during heat waves (heat stress). Two sorghum lines, with different leaf width were grown in a glasshouse at a mean day temperature of 30 °C, under different [CO2] and watering levels, and subjected to heat stress (43 °C) for 6 d at the start of the reproductive stage. We measured leaf photosynthetic and stomatal responses to light transients before harvesting the plants. Photosynthesis at growth conditions (Agrowth) and biomass accumulation were enhanced by eCO2 under control conditions. Heat stress increased gs, especially in wider leaves, and reduced the time constant of stomatal opening (kopen) at ambient [CO2] but not eCO2. However, heat stress reduced photosynthesis under water stress and eCO2 due to increased leaf temperature and reduced evaporative cooling. eCO2 prevented the reduction of biomass under both water and heat stress, possibly due to improved plant and soil water status as a result of reduced gs. Our results suggest that the response of the C4 crop sorghum to future climate conditions depends on the trade-off between low gs needed for high water use efficiency and drought tolerance, and the high gs needed for improved thermoregulation and heat tolerance under an eCO2 future.

The role of chloroplast movement in C4 photosynthesis: a theoretical analysis using a three-dimensional reaction-diffusion model for maize.

Chloroplasts movement within mesophyll cells in C4 plants is hypothesized to enhance the CO2 concentrating mechanism, but this is difficult to verify experimentally. A three-dimensional (3D) leaf model can help analyse how chloroplast movement influences the operation of the CO2 concentrating mechanism. The first volumetric reaction-diffusion model of C4 photosynthesis that incorporates detailed 3D leaf anatomy, light propagation, ATP and NADPH production, and CO2, O2 and bicarbonate concentration driven by diffusional and assimilation/emission processes was developed. It was implemented for maize leaves to simulate various chloroplast movement scenarios within mesophyll cells: the movement of all mesophyll chloroplasts towards bundle sheath cells (aggregative movement) and movement of only those of interveinal mesophyll cells towards bundle sheath cells (avoidance movement). Light absorbed by bundle sheath chloroplasts relative to mesophyll chloroplasts increased in both cases. Avoidance movement decreased light absorption by mesophyll chloroplasts considerably. Consequently, total ATP and NADPH production and net photosynthetic rate increased for aggregative movement and decreased for avoidance movement compared with the default case of no chloroplast movement at high light intensities. Leakiness increased in both chloroplast movement scenarios due to the imbalance in energy production and demand in mesophyll and bundle sheath cells. These results suggest the need to design strategies for coordinated increases in electron transport and Rubisco activities for an efficient CO2 concentrating mechanism at very high light intensities.

HUS with mutations in CFH and STEC infection treated with eculizumab in a 4-year-old girl.

BACKGROUND: Hemolytic uremic syndrome secondary to Shiga-toxin-producing Escherichia coli infection (STEC-HUS) generally shows a favorable outcome. Few cases develop extra-renal complications, since neurological involvement is an important cause of morbidity and mortality. The role of complement in STEC-HUS has been recently highlighted, and the use of eculizumab in severe cases has been communicated. HUS results from environmental and genetic factors, but the simultaneous occurrence of STEC and complement mutations remains undetermined. METHODS: A pediatric case with severe STEC-HUS carrying CFH mutations, with favorable response to eculizumab is analyzed. RESULTS: STEC-HUS was diagnosed in a 4-year-old girl with classic HUS, including low C3. Peritoneal dialysis was started due to hypertension, oligoanuria, and pleural effusion. She evolved with generalized tonic-clonic seizures and required mechanical ventilation. MRI reported multiple supra- and infratentorial ischemic lesions with laminar/striatal cortical necrosis and leukoencephalopathy. After two eculizumab doses, a significative stabilization in diuresis, blood pressure, creatinine, and C3 was achieved. At the third week, episodes of massive digestive bleeding and a life-threatening condition required a colectomy thus preserving the ileocecal valve. Due to atypical evolution, a genetic study was considered, identifying two heterozygous variants (CFH S1191L/V1197A). CONCLUSION: STEC-HUS in patients with a genetic predisposition has been previously reported, but the low frequency of occurrence makes it a rare disease. As in the present case, patients with atypical course might benefit from genetic analysis to evaluate early eculizumab initiation and to better understand its phenotype. A higher resolution version of the Graphical abstract is available as Supplementary information.

Lessons learned from regional training of paediatric nephrology fellows in Africa.

BACKGROUND: Access to care for children with kidney disease is limited in less well-resourced regions of the world and paediatric nephrology (PN) workforce development with good practical skills is critical. METHODS: Retrospective review of a PN training program and trainee feedback from 1999 to 2021, based at Red Cross War Memorial Children's Hospital (RCWMCH), University of Cape Town. RESULTS: A regionally appropriate 1-2-year training program enrolled 38 fellows with an initial 100% return rate to their country of origin. Program funding included fellowships from the International Pediatric Nephrology Association (IPNA), International Society of Nephrology (ISN), International Society of Peritoneal Dialysis (ISPD), and the African Paediatric Fellowship Program (APFP). Fellows were trained on both in- and out-patient management of infants and children with kidney disorders. "Hands-on skills" training included examination, diagnosis and management skills, practical insertion of peritoneal dialysis catheters for management of acute kidney injury and kidney biopsies. Of 16 trainees who completed > 1 year of training, 14 (88%) successfully completed subspecialty exams and 9 (56%) completed a master's degree with a research component. PN fellows reported that their training was appropriate and enabled them to make a difference in their respective communities. CONCLUSIONS: This training program has successfully equipped African physicians with the requisite knowledge and skills to provide PN services in resource-constrained areas for children with kidney disease. The provision of funding from multiple organizations committed to paediatric kidney disease has contributed to the success of the program, along with the fellows' commitment to build PN healthcare capacity in Africa. A higher resolution version of the Graphical abstract is available as Supplementary information.

IPNA clinical practice recommendations for the diagnosis and management of children with steroid-sensitive nephrotic syndrome.

Idiopathic nephrotic syndrome is the most frequent pediatric glomerular disease, affecting from 1.15 to 16.9 per 100,000 children per year globally. It is characterized by massive proteinuria, hypoalbuminemia, and/or concomitant edema. Approximately 85-90% of patients attain complete remission of proteinuria within 4-6 weeks of treatment with glucocorticoids, and therefore, have steroid-sensitive nephrotic syndrome (SSNS). Among those patients who are steroid sensitive, 70-80% will have at least one relapse during follow-up, and up to 50% of these patients will experience frequent relapses or become dependent on glucocorticoids to maintain remission. The dose and duration of steroid treatment to prolong time between relapses remains a subject of much debate, and patients continue to experience a high prevalence of steroid-related morbidity. Various steroid-sparing immunosuppressive drugs have been used in clinical practice; however, there is marked practice variation in the selection of these drugs and timing of their introduction during the course of the disease. Therefore, international evidence-based clinical practice recommendations (CPRs) are needed to guide clinical practice and reduce practice variation. The International Pediatric Nephrology Association (IPNA) convened a team of experts including pediatric nephrologists, an adult nephrologist, and a patient representative to develop comprehensive CPRs on the diagnosis and management of SSNS in children. After performing a systematic literature review on 12 clinically relevant PICO (Patient or Population covered, Intervention, Comparator, Outcome) questions, recommendations were formulated and formally graded at several virtual consensus meetings. New definitions for treatment outcomes to help guide change of therapy and recommendations for important research questions are given.

Functional Implications and Clinical Potential of MicroRNAs in Irritable Bowel Syndrome: A Concise Review.

MicroRNAs (miRNAs) are tiny (20-24 nucleotides long), non-coding, highly conserved RNA molecules that play a crucial role within the post-transcriptional regulation of gene expression via sequence-specific mechanisms. Since the miRNA transcriptome is involved in multiple molecular processes needed for cellular homeostasis, its altered expression can trigger the development and progression of several human pathologies. In this context, over the last few years, several relevant studies have demonstrated that dysregulated miRNAs affect a wide range of molecular mechanisms associated with irritable bowel syndrome (IBS), a common gastrointestinal disorder. For instance, abnormal miRNA expression in IBS patients is related to the alteration of intestinal permeability, visceral hyperalgesia, inflammatory pathways, and pain sensitivity. Besides, specific miRNAs are differentially expressed in the different subtypes of IBS, and therefore, they might be used as biomarkers for precise diagnosis of these pathological conditions. Accordingly, miRNAs have noteworthy potential as theragnostic targets for IBS. Hence, in this current review, we present an overview of the recent discoveries regarding the clinical relevance of miRNAs in IBS, which might be useful in the future for the development of miRNA-based drugs against this disorder.

Coordination of stomata and vein patterns with leaf width underpins water-use efficiency in a C4 crop.

Despite its importance for crop water use and productivity, especially in drought-affected environments, the underlying mechanisms of variation in intrinsic water-use efficiency (iWUE = net photosynthesis/stomatal conductance for water vapour, gsw ) are not well understood, especially in C4 plants. Recently, we discovered that leaf width (LW) correlated negatively with iWUE and positively with gsw across several C4 grasses. Here, we confirmed these relationships within 48 field-grown genotypes differing in LW in Sorghum bicolor, a C4 crop adapted to dry and hot conditions. We measured leaf gas exchange and modelled leaf energy balance three times a day, alongside anatomical traits as potential predictors of iWUE. LW correlated negatively with iWUE and stomatal density, but positively with gsw , interveinal distance of longitudinal veins, and the percentage of stomatal aperture relative to maximum. Energy balance modelling showed that wider leaves needed to open their stomata more to generate a more negative leaf-to-air temperature difference, especially at midday when air temperatures exceeded 40°C. These results highlight the important role that LW plays in shaping iWUE through coordination of vein and stomatal traits and by affecting stomatal aperture. Therefore, LW could be used as a predictor of higher iWUE among sorghum genotypes.

Phytochemicals mediated modulation of microRNAs and long non-coding RNAs in cancer prevention and therapy.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are two main categories of noncoding RNAs (ncRNAs) that can influence essential biological functions in various ways, as well as their expression and function are tightly regulated in physiological homeostasis. Additionally, the dysregulation of these ncRNAs seems to be crucial to the pathogenesis of human diseases. The latest findings indicate that ncRNAs execute vital roles in cancer initiation and progression, and the cancer phenotype can be reversed by modulating their expression. Available scientific discoveries suggest that phytochemicals such as polyphenols, alkaloids, terpenoids, and organosulfur compounds can significantly modulate multiple cancer-associated miRNAs and lncRNAs, thereby inhibiting cancer initiation and development. However, despite promising outcomes of experimental research, only a few clinical trials are currently being conducted to evaluate the therapeutic effectiveness of these compounds. Nevertheless, understanding phytochemical-mediated ncRNA regulation in cancer and the underlying molecular mechanisms on tumor pathophysiology can aid in the development of novel therapeutic strategies to combat this deadly disease.

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.

Endometrial area of the blood flow as a marker of endometritis in equine.

In this study, uterine blood flow area (BFA) has been evaluated for the first time using power Doppler ultrasound (PD) as a marker of endometritis in mares and jennies. The uterine BFA in healthy mares was greater in oestrus than in diestrus (p < .001). However, differences in endometrial blood flow between oestrus and diestrus were not observed in mares with endometritis. The uterine blood flow in healthy jennies is not affected by the oestrus cycle. Both species showed an increase in endometrial BFA in pathological uterine conditions compared to controls. BFA was a good marker of endometritis with an area under curve (AUC) (estrus:0.94 (p < .001) diestrus:0.98 (p < .001) in mares and AUC (0.91 (p < .0001) in jennies. The results of this preliminary study suggest that PD ultrasound in combination with computerized image analysis has the potential to be a very useful tool in the diagnosis of endometritis.

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.