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

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

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

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

Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation

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

Dataset of endometrial blood flow from pregnant and non-pregnant mares on day 7 and 8 post-ovulation

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

Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility

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

Power Doppler can detect the presence of 7–8 day conceptuses prior to flushing in an equine embryo transfer program

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 mm2 or two embryos: 61.01 ± 6.73 mm2) prior to embryo recovery compared to barren mares (21.77 ± 2.22 mm2) (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 mm2. 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 mm2 on day 7 (with a sensitivity of 75% and a specificity of 87.5%) and greater than 21.02 mm2 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 mm2 on day 7 (with a sensitivity of 80% and a specificity of 85.5%) and greater than 35.55 mm2 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 mm2) than young (17.21 ± 1.26 mm2) and middle aged non-pregnant mares (23.84 ± 1.50 mm2) (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

Membrane shape as a reporter for applied forces

Recent advances have enabled 3-dimensional reconstructions of biological structures in vivo, ranging in size and complexity from single proteins to multicellular structures. In particular, tomography and confocal microscopy have been exploited to capture detailed 3-dimensional conformations of membranes in cellular processes ranging from viral budding and organelle maintenance to phagocytosis. Despite the wealth of membrane structures available, there is as yet no generic, quantitative method for their interpretation. We propose that by modeling these observed biomembrane shapes as fluid lipid bilayers in mechanical equilibrium, the externally applied forces as well as the pressure, tension, and spontaneous curvature can be computed directly from the shape alone. To illustrate the potential power of this technique, we apply an axial force with optical tweezers to vesicles and explicitly demonstrate that the applied force is equal to the force computed from the membrane conformation

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