Search

Background: In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed., Results: IVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise, secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs., Conclusions: The addition of secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA)., (© 2024. The Author(s).)

The Mitochondrial distribution pattern or MDP in mammalian oocytes serves as an indicator of their cytoplasmic maturity, with a heterogeneous pattern associated with mature cytoplasm. Currently, MDP assessment involves fluorescent labelling of mitochondria followed by visual evaluation, as no quantitative method exists. Our objective was to develop a quantitative approach to assess MDP in mature equine oocytes. Equine oocytes, obtained by ovum pick up (OPU) were matured in vitro, and only metaphase II oocytes were used in the study (n = 56). Following denudation, oocytes were fixed, stained with MitoTracker™ Red CMXRos (50 nM in TCM-199 with Hank´s salts and 10% FBS) for 15 min at 38 °C, and then incubated with 2.5 µg/ml Hoechst 33342 for 10 min at 38 °C. Confocal microscope images were acquired, and the oocyte's MDP was visually classified as either homogeneous (HoD; n = 17) or heterogeneous (HeD; n = 39). For quantitative analysis, Fiji-ImageJ software was employed. Background subtraction was performed, and a 1-pixel line along the diameter was drawn to calculate the intensity profile. Fluorescence intensities were normalized, and ratios of peripheral to central fluorescence intensity were determined. Student´s t-test was used for comparations; MDP ratio was (mean ± standard error of the mean): 0.8 ± 0.02 for HoD and 0.3 ± 0.02 for HeD (p < 0.001). These results demonstrate concordance between quantitative and qualitative MDP assessment in mature equine oocytes. Our study describes a new approach to quantify mitochondrial distribution pattern and cytoplasmic maturation in mature equine oocytes., (© 2024. The Author(s).)

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2022 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

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"., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2022 The Author(s). Published by Elsevier Inc.)

Significance: Proper functionality of the spermatozoa depends on the tight regulation of their redox status; at the same time these cells are highly energy demanding and in the energetic metabolism, principally in the electron transport chain in the mitochondria, reactive oxygen species are continuously produced, in addition to that observed in the Krebs cycle and during the β-oxidation of fatty acids. Recent Advances: In addition, in glycolysis, elimination of phosphate groups from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate results in the byproducts glyoxal (G) and methylglyoxal (MG); these products are 2-oxoaldehydes. The presence of adjacent carbonyl groups makes them strong electrophiles that react with nucleophiles in proteins, lipids, and DNA, forming advanced glycation end products. Critical Issues: This mechanism is behind subfertility in diabetic patients; in the animal breeding industry, commercial extenders for stallion semen contain a supraphysiological concentration of glucose that promotes MG production, constituting a potential model of interest. Future Directions: Increasing our knowledge of sperm metabolism and its interactions with redox regulation may improve current sperm technologies in use, and shall provide new clues to understanding infertility in males. Moreover, stallion spermatozoa due to its accessibility, intense metabolism, and suitability for proteomics/metabolomic studies may constitute a suitable model for studying regulation of metabolism and interactions between metabolism and redox homeostasis. Antioxid. Redox Signal. 37, 521-537.

This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n = 33) were assessed using proven diagnostic techniques (spermiogram, B-mode and Pulse Doppler ultrasound), and subsequent analysis with Ecotext. Animals were classified as "control stallions" (n:21, acceptable semen quality), and "stallions with TD" (n:12, poor semen quality (TM < 60%, PM < 45% and total nº of sperm with PM < 2000 × 10 6 spz), that were subdivided into "induced TD group" (immunized, anti-GnRH vaccine) and "acquired TD group". The acquired TD group showed differences in all Ecotext parameters in relation to controls (Ecotext 1:0.11 ± 0.17 vs 2.82 ± 2.52, Ecotext 2:1584.0 ± 575.8 vs 388 ± 368.2, Ecotext 3:134.2 ± 9.26; ETA: 2.14 ± 0.59 vs 5.40 ± 1.90; ETd: 65.66 ± 6.27 vs 86.93 ± 10.65 and ETD: 92.35 ± 11.24 vs 132.10 ± 16.35, p ≤ 0.001). Results suggest acquired TD stallions were suffering testicular degeneration with loss of architecture and function as all Ecotext parameters were altered in relation to controls. Induced TD horses only showed a reduction in ETD (116.2 ± 8.59 vs 132.10 ± 16.35, p ≤ 0.001), despite all sperm parameters being worse. These findings suggested immunized stallions probably only experience an acute loss of testicular functionality and parenchyma architecture is likely not affected since differences in Ecotext parameters with control stallions were not detected. ETD was the best parameter to identify animals with TD (AUC: 0.84, optimal cut-off value of 124.3 seminiferous tubules/cm 2 ). Correlations were found between ETD and Doppler indices (PI: 0.60; RI: 0.47 p ≤ 0.001), total testicular volume (r: 0.48; p ≤ 0.05) and sperm motility (TM:0.51; and PM:0.54; p ≤ 0.001) and production (r:0.51; p ≤ 0.001). In summary, Ecotext could identify changes in testicular echotexture of stallions with TD. Results open the possibility for new research focused on establishing the relationship between Ecotext parameters and histomorphometry features in stallion testes., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest that could be perceived to prejudice the reported research., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. SIGNIFICANCE: Proteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This paper provides the dataset of proteins of stallion ejaculates before and after cryopreservation. The data report the analysis and identification of stallion sperm proteins obtained from the same ejaculates and split in two subsamples. The first aliquot consisted on fresh spermatozoa and the second aliquot was frozen and thawed spermatozoa. Samples were analyzed using a UHPLC/MS/MS system consisting of an Agilent 1290 infinity series UHPLC coupled to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). We provide a data set of 2226 different proteins, with 2180 aligned to the equine proteome database. The data can be used to identify potential targets to be explored to improve techniques for cryopreservation of spermatozoa. This data article refers to the article "Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation" (Martín Cano et al.; 2020) [1]., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Author(s).)

Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/MS/MS was used to study the sperm proteome under these two distinct conditions and bioinformatic enrichment analysis conducted. Gene Ontology (GO) and pathway enrichment analysis were performed revealing dramatic changes as consequence of cryopreservation. The terms oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport and electron transport chain were significantly enriched in fresh samples (P = 5.50 × 10 -12 , 4.26 × 10 -8 and 7.26 × 10 -8, respectively), while were not significantly enriched in frozen thawed samples (P = 1). The GO terms oxidation reduction process and oxidoreductase activity were enriched in fresh samples and the enrichment was reduced in frozen thawed samples (1.40 × 10 -8 , 1.69 × 10 -6 versus 1.13 × 10 -2 and 2-86 × 10 -2 respectively). Reactome pathways (using human orthologs) significantly enriched in fresh sperm were TCA cycle and respiratory electron transport (P = 1.867 × 10 -8 ), Respiratory electron transport ATP synthesis by chemiosmosis coupling (P = 2.124 × 10 -5 ), Citric acid cycle (TCA cycle)(P = 8.395 × 10 -4 ) Pyruvate metabolism and TCA cycle (P = 3.380 × 10 -3 ), Respiratory electron transport (P = 2.764 × 10 -2 ) and Beta oxidation of laurolyl-CoA to decanoyl CoA-CoA (P = 1.854 × 10 -2 ) none of these pathways were enriched in thawed samples (P = 1). We have provided the first detailed study on how the cryopreservation process impacts the stallion sperm proteome. Our findings identify the metabolic proteome and redoxome as the two key groups of proteins affected by the procedure. SIGNIFICANCE: In the present manuscript we investigated how the cryopreservation of stallion spermatozoa impacts the proteome of these cells. This procedure is routinely used in horse breeding and has a major impact in the industry, facilitating the trade of genetic material. This is still a suboptimal biotechnology, with numerous unresolved problems. The limited knowledge of the molecular insults occurring during cryopreservation is behind these problems. The application and development of proteomics to the spermatozoa, allow to obtain valuable information of the specific mechanisms affected by the procedure. In this paper, we report that cryopreservation impacts numerous proteins involved in metabolism regulation (mainly mitochondrial proteins involved in the TCA cycle, and oxidative phosphorylation) and also affects proteins with oxidoreductase activity. Moreover, specific proteins involved in the sperm-oocyte interaction are also affected by the procedure. The information gathered in this study, opens interesting questions and offer new lines of research for the improvement of the technology focusing the targets here identified, and the specific steps in the procedure (cooling, toxicity of antioxidants etc.) to be modified to reduce the damage., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest that could be perceived to prejudice the reported research., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This article provides the dataset for the use of power Doppler ultrasound to assess the equine uterus from the recent research article titled "Power Doppler can detect the presence of 7-8 days conceptuses prior to flushing in an equine embryo transfer program"(1). The vascularization of the endometrium was objectively assessed in mares by quantification of pixels in bitmap format (BMP) using computer assisted analysis of images. Fifty-two mares were examined on days 7 (26 mares) and 8 (26 mares) post-ovulation prior to performing flushing procedures for embryo recovery. Receiver operating characteristic (ROC) curves and Youden's J statistics were used to evaluate the value of the suggested variable in terms of its diagnostic value for identification of early pregnancy and to establish cut-off values allowing differentiation between pregnant and non-pregnant mares on days 7 and 8 post-ovulation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Authors.)

In order to determine whether differences in uterine blood flow between pregnant and non-pregnant mares can be used to predict the presence of the equine embryo prior to flushing in an embryo transfer program, power Doppler ultrasonography was used on a total of 52 mares on days 7 or 8 post-ovulation. Computer analysis of Doppler images was subsequently performed using ImageJ v1.48 software. Vascular perfusion of the endometrium was analyzed using spot meter techniques, measuring mean pixel intensity and area of blood flow. Mares with positive flushings presented a higher uterine blood flow area (one embryo: 54.01 ± 2.27 mm 2 or two embryos: 61.01 ± 6.73 mm 2 ) prior to embryo recovery compared to barren mares (21.77 ± 2.22 mm 2 ) (p≤0.05). However, significant differences in vascular perfusion were not detected between single or twin pregnancies. Blood flow area appears to be a good predictor for differentiation between pregnant and non-pregnant mares with an AUC: 0.869; p≤0.001 and an optimal cut-off value of 37.21 mm 2 . Both the mare's age and day of embryo recovery caused effects on uterine vascular perfusion. According to Youden's J statistics the uterine blood flow area of young pregnant mares was greater than 25.4 mm 2 on day 7 (with a sensitivity of 75% and a specificity of 87.5%) and greater than 21.02 mm 2 on day 8 post-ovulation (with a sensitivity of 93.8% and a specificity of 100%). The uterine blood flow area in adult pregnant mares was greater than 41.4 mm 2 on day 7 (with a sensitivity of 80% and a specificity of 85.5%) and greater than 35.55 mm 2 on day 8 after ovulation (with a sensitivity of 97.2% and a specificity of 85.7%). Evaluation on day 8 is therefore considered to be more reliable. Older and middle aged pregnant mares (5-18 years old) had increased uterine vascularization compared to young pregnant mares (2-5 years old) (p≤0.001). Conversely, older barren mares showed higher endometrial vascularity (35.06 ± 2.56 mm 2 ) than young (17.21 ± 1.26 mm 2 ) and middle aged non-pregnant mares (23.84 ± 1.50 mm 2 ) (p≤0.05). We hypothesized that the higher blood flow area seen in older barren mares may be a consequence of a subclinical endometritis due to repeated flushing for embryo recovery. The results of the present study indicate that power Doppler ultrasound combined with computer assisted analysis of images are reliable techniques to detect early pregnancy prior to embryo recovery., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine residues of key proteins acting as an "on-off" switch controlling sperm function. However, if deregulation occurs, these residues may experience irreversible oxidation and oxidative stress, leading to malfunction and ultimately death of the spermatozoa. Stallion spermatozoa are "professional producers" of reactive oxygen species due to their intense mitochondrial activity, and thus sophisticated systems to control redox homeostasis are also characteristic of the spermatozoa in the horse. As a result, and combined with the fact that embryos can easily be collected in this species, horses are a good model for the study of redox biology in the spermatozoa and its impact on the embryo.

Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, and C. Single layer centrifugation through Androcoll-E (SLC). All samples were preserved in INRA 96 at 5 °C for 72 h. Aliquots from native semen and from different treatments were taken for bacteriological analysis at T0, T24, T48 and T72h of storage and Total microbial load (TML: CFU (colony-forming units/ml) was calculated. The ROS production (dichlorodihydrofluorescein diacetate for H 2 O 2 , dihydroethidium for superoxide anion and CellROX deep red for total ROS), viability (YO-PRO-1-Ethidium) and lipid peroxidation (BODIPY-C11) were assessed by flow cytometry, and motility by CASA. The bacteria isolated were Corynebacterium spp, Arcanobacterium spp, Bacillus spp, Dermobacter, Staphylococcus spp, Streptococcus spp, Penicilium spp. TML of semen showed correlations with live sperm (r: -0.771), dead sperm (r: 0.580), H 2 O 2 production (r: 0.740), and total ROS production (CellROX (+)) (r: -0.607), Total motility (r: 0.587), Progressive motility (r: -0.566), VCL (r: -0.664), VSL (r: -0,569), VAP (r: -0.534) (p ≤ 0.05). SLC removed 99.34% of the microbial load, which was assicated with a significanlty reduced H 2 O 2 production (p ≤ 0.05). However, only samples treated with Androcoll-E had a higher total ROS production (CellROX +) (p ≤ 0.05). These results suggest that CellROX stain probably identifies superoxide production rather than H 2 O 2 and this higher superoxide production may reflect an intense sperm functionality. The bacterial load increased the production of H 2 O 2 in cool-stored semen which was associated with lower tolerance to refrigeration. SLC was the sperm processing technique that was most efficient at removing bacteria, reducing H 2 O 2 production and selecting the most functional sperm., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 FeSO 4 exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8-iso-PGF 2α 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., (© 2017 Blackwell Verlag GmbH.)

A multiscale electrothermal simulation approach is presented to optimize the design of a hybrid switch soft-switching inverter using a library of dynamic electrothermal component models parameterized in terms of electrical, structural, and material properties. Individual device area, snubber capacitor, and gate drive timing are used to minimize the total loss of the soft-switching inverter module subject to the design constraints including total device area and minimum on-time consideration. The proposed multiscale electrothermal simulation approach allows for a large number of parametric studies involving multiple design variables to be considered, drastically reducing simulation time. The optimized design is then compared and contrasted with an already existing design from the Virginia Tech Freedom Car Project using the generation II module. It will be shown that the proposed approach improves the baseline design by 16% in loss and reduces the cooling requirements by 42%. Validation of the electrical and thermal device models against measured data is also provided.

The most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. Novel methods based on Artificial Neural Networks have been widely investigated. In prior works, back propagation neural networks (BPNN) have been used to solve the neutron spectrometry problem, however, some drawbacks still exist using this kind of neural nets, i.e. the optimum selection of the network topology and the long training time. Compared to BPNN, it's usually much faster to train a generalized regression neural network (GRNN). That's mainly because spread constant is the only parameter used in GRNN. Another feature is that the network will converge to a global minimum, provided that the optimal values of spread has been determined and that the dataset adequately represents the problem space. In addition, GRNN are often more accurate than BPNN in the prediction. These characteristics make GRNNs to be of great interest in the neutron spectrometry domain. This work presents a computational tool based on GRNN capable to solve the neutron spectrometry problem. This computational code, automates the pre-processing, training and testing stages using a k-fold cross validation of 3 folds, the statistical analysis and the post-processing of the information, using 7 Bonner spheres rate counts as only entrance data. The code was designed for a Bonner Spheres System based on a 6 LiI(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The process of unfolding the neutron energy spectrum has been subject of research for many years. Monte Carlo, iterative methods, the bayesian theory, the principle of maximum entropy are some of the methods used. The drawbacks associated with traditional unfolding procedures have motivated the research of complementary approaches. Back Propagation Neural Networks (BPNN), have been applied with success in neutron spectrometry and dosimetry domains, however, the structure and learning parameters are factors that highly impact in the networks performance. In ANN domain, Generalized Regression Neural Network (GRNN) is one of the simplest neural networks in term of network architecture and learning algorithm. The learning is instantaneous, requiring no time for training. Opposite to BPNN, a GRNN would be formed instantly with just a 1-pass training on the development data. In the network development phase, the only hurdle is to optimize the hyper-parameter, which is known as sigma, governing the smoothness of the network. The aim of this work was to compare the performance of BPNN and GRNN in the solution of the neutron spectrometry problem. From results obtained it can be observed that despite the very similar results, GRNN performs better than BPNN., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

NSDUAZ (Neutron Spectrometry and Dosimetry from the Universidad Autónoma de Zacatecas) is a user friendly neutron unfolding package for Bonner sphere spectrometer with (6)LiI(Eu) developed under LabView(®) environment. Unfolding is carried out using a recursive iterative procedure with the SPUNIT algorithm, where the starting spectrum is obtained from a library initial guess spectra to start the iterations. The NSDUAZ performance was evaluated using (252)Cf, (252)Cf/D(2)O, (241)AmBe neutron sources and the neutrons outside the radial beam port of a TRIGA Mark III nuclear reactor running to 10 W., (Copyright © 2012 Elsevier Ltd. All rights reserved.)