Boar semen quality changes throughout a year and field fertility predictable value of sperm evaluation lab assays

The purpose of the present thesis was to study the characteristics of seminal parameters, and the expression of selected spermatozoa’s and seminal plasma proteins during a year and to identify their possible relationship to field fertility. Sixty-five (65) seminal samples were analyzed for the following parameters: viability, motility, kinetics, morphology, biochemical activity and functional ability of sperm membranes (HOST test), sperm mitochondrial potential and chromatin integrity. The evaluated seminal samples were collected from 18 boars, on a weekly basis for a whole year, while 468 sows were inseminated. The second experimental part determined the concentration of selected proteins of both sperm and seminal plasma. The third part of the thesis concerned the selection of the appropriate statistical analysis models, regarding the correlation of proteins’ concentration and sperm parameters with field fertility, as well as the investigation of possible seasonal changes in their values. Moreover, the impact of multiple sperm quality and protein parameters’ effects on swine AI’s outcome was studied. The first part of the experiments revealed that the percentage of immotile spermatozoa explains 6.7% (p = 0.04) of the variation of live born piglets and 6.5% (p = 0.04) of the variation of births with ≥12 piglets. The percentage of HOST-positive spermatozoa interprets 24.5% (p = 0.0001) of the live born piglets and 7.8% of the farrowings with ≥12 piglets. The variation of live born piglets can also be explained by the percentage of activated mitochondria (p = 0.01) by 13.5%. Finally, head morphological abnormalities can explain the variation of live born piglets by 8.9% (p= 0.02). Based on the data obtained from the second part of the experiments, there is a strong relation of seminal plasma osteopontin (OPN) with births with ≥12 piglets. Particularly, OPN70 and OPN12 explain the variation of births with ≥12 piglets by 6.5% (p = 0.04), and 8.8% (p = 0.02), respectively. In addition, the GPX5 sperm protein explains 6.7% (p = 0.04) of the farrowing rate. In terms of the seasonal variation of the examined parameters, significantly higher values of VSL, LIN (p = 0.04 and p = 0.03, respectively) and a lower percentage of sperm neck abnormalities (p = 0.01), where found in cold (October to June) compared to the warm (July to September) season. Concerning the proteins, a strong trend was observed with higher values of HSP90 and GPX5 in warm compared to the cold season (p = 0.07 and p = 0.06, respectively). In conclusion a) HOST is the most reliable assay for interpreting the variation of live born piglets; b) the variation of births with ≥12 piglets can be explained by the percentage of immotile sperm, the percentage of spermatozoa with active mitochondria and the concentration of OPN70 and OPN12; c) the GPX5 enzyme explains to a mean level the farrowing rate; d) the warm period negatively affects VSL and LIN, while the concentrations and hence the protective activity of GPX5 and HSP90 proteins increased significantly over the same period.Σκοπός της παρούσας διατριβής ήταν η μελέτη μιας σειράς παραμέτρων του σπέρματος, καθώς και επιλεγμένων πρωτεϊνών των σπερματοζωαρίων και του σπερματικού πλάσματος κατά τη διάρκεια ενός έτους, ώστε να εντοπισθούν οι πιθανές σχέσεις των μεταβολών αυτών με παραμέτρους γονιμότητας σε συνθήκες πράξης. Το πρώτο σκέλος της διατριβής είχε ως βασικό σκοπό την ανάλυση 65 δειγμάτων σπέρματος κάπρου ως προς τις παραμέτρους: ζωτικότητα, κινητικότητα, επί μέρους μορφές κίνησης, μορφολογία, βιοχημική δραστηριότητα-λειτουργική ικανότητα των μεμβρανών των σπερματοζωαρίων (δοκιμή HOST), λειτουργικότητα των μιτοχονδρίων και ακεραιότητα της χρωματίνης του πυρήνα των σπερματοζωαρίων. Τα δείγματα σπέρματος συλλέχθηκαν, σε εβδομαδιαία βάση, στη διάρκεια του έτους από 18 σπερματοδότες κάπρους. Τα εκσπερματίσματα χρησιμοποιήθηκαν για τη σπερματέγχυση 468 συών της ίδιας εκτροφής. Στο δεύτερο σκέλος προσδιορίστηκε η συγκέντρωση επιλεγμένων πρωτεϊνών τόσο των σπερματοζωαρίων όσο και του σπερματικού πλάσματος. Αρχικά, διαπιστώθηκε ότι κάποιες από τις παραμέτρους του σπέρματος που εξετάστηκαν σχετίζονταν σημαντικά με ορισμένους από τους δείκτες γονιμότητας. Το ποσοστό των ακίνητων σπερματοζωαρίων επηρέασε σημαντικά (p=0,04) τον αριθμό των ζωντανών γεννηθέντων χοιριδίων, εξηγώντας κατά 6,7% τη μεταβλητότητά τους, αλλά και τους τοκετούς με τοκετοομάδες ≥12 χοιριδίων (r2=0,065, p=0,04). Παράλληλα, και το ποσοστό των σπερματοζωαρίων με μορφολογικές ανωμαλίες της κεφαλής επηρέασε ακόμη περισσότερο (p=0,02) τον αριθμό των ζωντανών γεννηθέντων χοιριδίων, εξηγώντας κατά 8,9% τη διακύμανσή τους. Από την άλλη πλευρά το ποσοστό των σπερματοζωαρίων που ανταποκρίνονταν θετικά στη δοκιμή HOST επηρέασε πολύ σημαντικά (p=0,0001) τις παραπάνω παραμέτρους γονιμότητας εξηγώντας κατά 24,5% τη μεταβλητότητα του αριθμού των ζωντανών γεννηθέντων χοιριδίων και κατά 7,8% τη μεταβλητότητα των τοκετών με ≥12 χοιρίδια. Ομοίως και το ποσοστό των σπερματοζωαρίων με δραστηριοποιημένα μιτοχόνδρια επηρέασε πολύ σημαντικά (p=0,01) τον αριθμό των ζωντανών γεννηθέντων χοιριδίων εξηγώντας αντίστοιχα κατά 13,5% τη μεταβλητότητά τους. Αναφορικά με τις πρωτεΐνες προέκυψε τόσο η πρωτεΐνη του σπερματικού πλάσματος οστεοποντίνη OPN70, όσο και η OPN12 επηρέασαν σημαντικά (p=0,04 και p=0,02, αντίστοιχα) τον αριθμό των τοκετών με τοκετοομάδες ≥12 χοιριδίων, εξηγώντας αντίστοιχα κατά 6,5% και 8,8% τη μεταβλητότητά τους. Επιπρόσθετα, η πρωτεΐνη GPX5 των σπερματοζωαρίων επηρέασε σημαντικά (p=0,04) το ποσοστό των τοκετών εξηγώντας κατά 6,7% τη μεταβλητότητά του. Μεταξύ των εκσπερματισμάτων που συλλέχθηκαν στη διάρκεια της «ψυχρής» και της «θερμής» περιόδου, διαπιστώθηκαν σημαντικά μεγαλύτερες τιμές των παραμέτρων κινητικότητας των σπερματοζωαρίων VSL και LIN (p=0,04 και p=0,03, αντίστοιχα), καθώς και σημαντικά μικρότερο ποσοστό ανωμαλιών του αυχένα των σπερματοζωαρίων (p=0,01), την ψυχρή συγκριτικά με τη θερμή περίοδο. Αντίθετα, τη θερμή περίοδο προέκυψε ισχυρή τάση αύξησης των πρωτεϊνών HSP90 και GPX5 σε σύγκριση με την ψυχρή (p=0,07 και p=0,06, αντίστοιχα). Προέκυψαν τα ακόλουθα συμπεράσματα: α) η δοκιμή HOST μπορεί να χρησιμοποιηθεί για την ερμηνεία της μεταβλητότητας των ζωντανών γεννηθέντων χοιριδίων, β) τα ποσοστά των ακίνητων σπερματοζωαρίων και των σπερματοζωαρίων με ενεργοποιημένα μιτοχόνδρια καθώς και οι συγκεντρώσεις των πρωτεϊνών OPN70 και OPN12 στο σπερματικό πλάσμα, μπορούν να χρησιμοποιηθούν για την ερμηνεία της μεταβλητότητας των τοκετών με ≥12 χοιρίδια, γ) η GPX5 εξηγεί ως έναν βαθμό τη μεταβλητότητα του ποσοστού των τοκετών, δ) η θερμή περίοδος επηρεάζει περισσότερο αρνητικά τις παραμέτρους κινητικότητας των σπερματοζωαρίων VSL και LIN, ενώ οι συγκεντρώσεις των πρωτεϊνών GPX5 και HSP90 αυξάνονται στην ίδια περίοδο

Deliverable D8.1 UC specifications and demonstrator deployment plan

This report provides a detailed description of the Greek demonstration, its pilot site, its planned activities, its Use Cases and its evaluation plan. It is part of the activities of WP8 and concludes Task 8.1 “Demonstrator Specification and Planning and KPIs Definition”. It corresponds to similar deliverables from the other demonstrations (WP6, 7 and 9) and is related to deliverable D1.5 “Use Case Specifications” [1]. Moreover, it draws input from deliverable D5.3 “Open V2X Management Platform – Architecture” [2] which describes the O-V2X Platform, a key part of the Greek demo.The pilot site description focuses on analysing the characteristics of the chosen location which has been the pilot site in past projects, such as the Wisegrid [3], Platone [4] and SYNERGY [5] The local energy system of the demo location (Mesogia region of Attica) is in a semi-rural area and includes hundreds of thousands of customers, as well as medium (MV) and low voltage (LV) Photovoltaic (PV) installations. In addition, some customers have smart meters and there are several charging stations operated by project partners. Another highlight in the pilot site is the presence of GIS (Geographic Information System) technology that works in cooperation with the local DMS-SCADA (Distribution Management System - Supervisory Control and Data Acquisition) system. Recently, the site’s AMI (Advanced Metering Infrastructure) infrastructure has been augmented with PMUs (Phasor Measurement Unit) which support state-estimation algorithms.In the context of the Greek demonstration, the project involves three primary actors and four secondary actors. The primary actors are integral to the project's success, with HEDNO serving as the Distribution System Operator (DSO) responsible for monitoring the network, managing Distributed Energy Resources (DER) flexibility, and adjusting tariff prices to alleviate congestion. PPC fulfils the role of the Charging Point Operator (CPO), overseeing public charging stations, participating in flexible capacity contracts, and offering green charging services. The Platform Manager (PM) focuses on developing and maintaining the Open V2X Management Platform (O-V2X-MP), aimed at enhancing the electric vehicle ecosystem and streamlining charging infrastructure management and is undertaken also by PPC. In addition to these main actors, two other actors play essential roles: Citroen, representing the Electric Vehicle (EV) manufacturer, provides valuable insights into EV-related aspects, and EV users, who seek cost-effective and convenient charging solutions while interacting with the CPO's services and the PM's platform. Furthermore, two minor simulated roles, the flexibility market operator and consumers, are instrumental in simulating and overseeing transactions and diverse electrical loads within the project's dynamics.The Greek pilot project consists of several key phases. It begins with setting specifications for components, including the O-V2X-MP and DSO Platform developed by PPC and HEDNO, LV monitoring infrastructures, and charging point infrastructures, with a target completion date at the end of November 2023. Subsequent phases involve deploying and testing the O-V2X MP's communications with charging stations and EVs, integrating it with DSO systems, and testing information and signal exchange based on standards by the end of May 2024. The commission of the O-V2X-MP platform, integration of management algorithms, LV monitoring system integration with the DSO platform, and communication testing will occur by the end of November 2024. DSO and CPO will test flexible capacity contracts activation and Green Charging, with communication through O-V2X-MP between them, to be completed until the end of February 2025. The project will conclude with the evaluation of new business models, Vehicle to Grid (V2G) vs. Smart Charging (V1G) benefits, and the documentation of knowledge gained for exploitation and marketability strategies until the end of August 2025.There are two installations of systems and two deployments of tools that are planned by the Greek demo partners. Firstly, HEDNO is leading the installation of LV feeder supervision systems at MV/LV substations to enhance distribution network observability. The system includes measurement devices EV4EU – D8.1 UC specifications and demonstrator deployment plan for electrical parameters, data concentrators, and communication via cellular networks. Secondly, PPC is responsible for setting up 2 to 5 public EV charging points at various locations within the pilot site, complying with Greek regulations and standards. These charging stations support one-phase or three-phase charging and offer safety features, including ground fault monitoring. They also support user/billing management programs through the Open Charge Point Protocol (OCPP) and are upgradeable to OCPP 2.0 for future innovations in electric mobility, including vehicle to-grid (V2G) capabilities. In addition to field equipment installations, HEDNO's DSO Support System (DSS) serves as a communication hub connecting with CPO platforms, facilitating practical tests of Business Use Case 4 (BUC 4) and Business Use Case 5 (BUC 5), and improving power distribution network monitoring and resource optimization. Meanwhile, PPC's Open V2X platform acts as a central coordinator, promoting seamless interactions between electric vehicles, charging infrastructure, and the energy grid by offering various services such as CPO and E-Mobility Service Provider (EMSP) services, roaming services, and a billing engine for precise cost management. The platform employs Open Charge Point Interface (OCPI) services for data exchange and integrates Location, Charging Data Records (CDR), Tariff, and Customer Relationship Management (CRM) Application Programming Interfaces (APIs) for station information, session details, tariff flexibility, and user feedback.The demonstration plan consists of four phases. Phase A focuses on verifying the correct hardware installation of charging stations and LV metering equipment. Phase B emphasizes the verification of communication between various components, ensuring accurate message transmission and resilience against cyberattacks. Phase C aims to validate the cohesive operation of all components within the demo site and the functionality of developed algorithms. For Business Use Case 4 BUC4 (Green Charging), the DSO platform generates price signals based on PV energy forecasts, while for Business Use Case 5 (BUC5) (Flexible Capacity Contracts), the DSO forecasts and provides capacity limit signals to the CPO platform. Phase D involves incorporating the platform into DSO systems and conducting real-scale validation to address interoperability, user responsiveness, impact on distribution network security, and DSO flexibility management. Additionally, at least one V2G charging station will be installed for lab tests and simulations, including reverse power provision tests during the demonstration phase.Additionally, to the field and deployment activities, the Business Use Cases (BUCs) are described in detailed. Business Use Case 4 (BUC 4) demonstrates how electric vehicles (EVs) can enhance renewable energy integration. It aims to reduce grid constraints, lower medium voltage grid power injection, and delay distribution grid investments. BUC 4 employs Demand Response (DR) to coordinate renewable energy and EV activities. When reverse power flow occurs, the DSO identifies locations for price reductions, communicated to the CPO managing charging stations. The CPO adjusts prices, conveyed to EV owners via the Platform Manager, allowing them to optimize charging based on pricing changes. Two scenarios are tested, one at a single MV/LV substation and another on a broader MV network scale. The process involves issue detection, tariff reductions, communication to CPOs, and notifying EV users for efficient charging decisions. Business Use Case 5 (BUC 5) demonstrates the use of flexible capacity contracts in V2X scenarios, focusing on reducing distribution system constraints, delaying grid investments, and expanding CPO service offerings. In these scenarios, the DSO procures capacity limitation services from CPOs in a medium-term market, activating them either in advance or in real-time to address network congestion. The process includes DSO identification of congestion, communication with CPOs, adjustments of charging station capacity limits, communication with EV owners through the Platform Manager, and EV owners making charging decisions based on preferences, with feedback relayed to CPOs for prompt updates to charging station status.Finally, the evaluation plan of the Greek demo was created with its main feature being the demo KPIs which are described in detail and mapped to the BUCs. In total there are 15 Key Performance Indicators EV4EU – D8.1 UC specifications and demonstrator deployment plan (KPIs) covering a range of topics, namely: technical, economic and market, environmental and social, and service related

Comparative Effects of Deoxynivalenol, Zearalenone and Its Modified Forms De-Epoxy-Deoxynivalenol and Hydrolyzed Zearalenone on Boar Semen In Vitro

Deoxynivalenol (DON) and zearalenone (ZEN) are described as detrimental factors to sow and boar fertility. In comparison, literature reports on the impact of modified forms of DON and ZEN, such as de-epoxy-DON (DOM-1) and hydrolyzed ZEN (HZEN), on swine reproduction are scarce. The aim of our study was to compare the effects of DON, DOM-1, ZEN and HZEN on boar semen in vitro. To this end, pooled boar semen ejaculates from two adult boars were treated with either 50.6 μM DON, 62.8 μM ZEN or equimolar concentrations of DOM-1 and HZEN, respectively (dilution volume of v/v 0.7% DMSO in all cases). Effects on semen motility, morphology, viability, hypo-osmotic swelling test reaction and DNA integrity were investigated hourly up to four hours of incubation. DON negatively affected particular parameters evaluated with a computer-assisted sperm analysis system (CASA), such as immotile spermatozoa and progressive motile spermatozoa, whereas those effects were absent in the case of DOM-1 treatment. In contrast to HZEN, ZEN affected almost all CASA parameters. Furthermore, only ZEN decreased the proportion of viable spermatozoa and increased the proportion of spermatozoa with abnormalities. In conclusion, DON and ZEN negatively affected boar semen in vitro, whereas equimolar concentrations of DOM-1 and HZEN did not induce harmful effects

Effect of Boar Sperm Proteins and Quality Changes on Field Fertility

This study aimed to evaluate boar sperm characteristics and proteins, in relation to their importance regarding in vivo fertility. Sixty-five ejaculates were used and 468 sows (parity ≥ 2) were inseminated. Sperm CASA kinetics, morphology, viability, DNA fragmentation, mitochondrial membrane potential, sperm membrane biochemical activity (HOST) and sperm proteins (Heat Shock Protein 90-HSP90, glutathione peroxidase-5-GPX5, Osteopontin 70-OPN70) were assessed and related to field fertility (number of live-born piglets—NLBP, litter size ≥ 12 piglets—LS, farrowing rate—FR). Statistical analysis was conducted with simple and multiple regression models. Simple regression analysis showed that immotile sperm (IM) significantly affected the NLBP and LS, explaining 6.7% and 6.5% of their variation, respectively. The HOST positive spermatozoa significantly affected the NLBP and LS, explaining 24.5% and 7.8% of their variation, respectively. Similarly, sperm with activated mitochondria significantly affected the NLBP, explaining 13.5% of its variation. Moreover, the OPN70 affected LS and FR, explaining 7.5% and 10.8% of their variation, respectively. Sperm GPX5 protein affected FR, explaining 6.7% of its variation. Multiple regression analysis showed that the combination of IM and/OPN70 explains 13.0% of the variation regarding LS, and the combination of GPX5 and OPN70 explains 13.6% of the variation regarding FR. In conclusion, the estimation of parameters IM, membrane biochemical activity, mitochondrial membrane potential, OPN and GPX5 can provide useful information regarding semen doses for field fertility

Iron Oxide Nanoparticles as an Alternative to Antibiotics Additive on Extended Boar Semen

This study examined the effect of Fe3O4 nanoparticles on boar semen. Beltsville thawing solution without antibiotics was used to extend ejaculates from 5 boars (4 ejaculates/boar). Semen samples of control group (C) and group with Fe3O4 (Fe; 0.192 mg/mL semen) were incubated under routine boar semen storage temperature (17 °C) for 0.5 h and nanoparticles were removed by a magnetic field. Before and after treatment, aliquots of all groups were cultured using standard microbiological methods. The samples after treatment were stored (17 °C) for 48 h and sperm parameters (computer-assisted sperm analyzer (CASA) variables; morphology; viability; hypo-osmotic swelling test (HOST); DNA integrity) were evaluated at storage times 0, 24, 48 h. Semen data were analyzed by a repeated measures mixed model and microbial data with Student’s t-test for paired samples. Regarding CASA parameters, Fe group did not differ from C at any time point. In group C, total motility after 24 h and progressive motility after 48 h of storage decreased significantly compared to 0 h. In group Fe, linearity (LIN) after 48 h and head abnormalities after 24 h of storage increased significantly compared to 0 h. The microbiological results revealed a significant reduction of the bacterial load in group Fe compared to control at both 24 and 48 h. In conclusion, the use of Fe3O4 nanoparticles during semen processing provided a slight anti-microbiological effect with no adverse effects on sperm characteristics

Toxic and Microbiological Effects of Iron Oxide and Silver Nanoparticles as Additives on Extended Ram Semen

The aim of the study was to investigate the effect of iron oxide (Fe) and silver (Ag) nanoparticles (NPs) on ram semen. A skim milk extender without antibiotics was used as a diluent of 21 ejaculates (8 rams; 2–3 ejaculates/ram). The groups of control (C; semen without NPs), Fe NPs (3.072 mg Fe3O4/mL semen), and Ag NPs (2.048 mg Ag-Fe/mL semen) were incubated (15 °C; 30 min), and then a magnetic field was used for NPs’ removal. Standard microbiological procedures were performed for all groups. Post-treated samples were stored (15 °C) for 24 h, and sperm variables (kinetics by computer assisted sperm analysis (CASA); viability; morphology; HOST; DNA integrity) were evaluated at 6 and 24 h. Semen data were analyzed by a mixed model for repeated measures and microbiological data with Student’s t-test for paired samples. At 6 h of storage, VCL and rapid movement-spermatozoa, and at 24 h, total/progressive motility and amplitude of lateral head displacement (ALH) were significantly decreased in group Ag compared to control. In group Fe, progressive/rapid movement-spermatozoa were significantly lower compared to control after 24 h of storage. Only in group Ag was a significant reduction of total bacterial count revealed. In conclusion, the examined Fe NPs demonstrated slight antibacterial effect, while the examined Ag NPs provided higher antibacterial properties accompanied by cytotoxicity