VALIDATION OF CONFIRMATORY MULTI-RESIDUE METHOD FOR THE DETERMINATION OF THIRTEEN HORMONES RESIDUES IN SEVERAL BOVINES MATRICES USING HPLC-MS/MS

Synthetic and natural hormones are used in the production and intensive breeding of animals as growth factors and reproductive regulators. However, the illegal and inappropriate use of these substances increases the risk of introducing residues into the food chain. Also because of their carcinogenic and teratogenic effects, the European Union has banned the use of these substances since 1985. For this reason, the development of sensitive and reliable analytical methods for the monitoring of their residues in food has become a necessity. A liquid chromatography analysis method coupled to mass spectrometry and based on Quechers extraction has been developed to analyse thirteen synthetic and natural hormones in muscles. In order to study the reliability of this method, its validation has been carried out in different bovine matrices (liver, kidney, bile, and hair) according to European Decision 2002/657 / EC. The method demonstrates good linearity (R2> 0.99) as well as accuracy with coefficients of variation for repeatability and reproducibility less than 23%. The values of CCα and CCβ were determined for each analyte indicating values ranging from 0.13 to 0.86 μg.kg-1 and 0.25 to 1.72 μg.kg-1 respectively for most of the analytes. Higher values were obtained for 17β-estradiol, estriol, 17α-ethinyl-estradiol ranging from 1.73 to 13.95 μg.kg-1 for CCα and from 3.47 to 23.87 μg.kg-1 for CCβ. The recovery rate in the different matrices (liver, kidney, bile, and hair) varies from 51.5 to 107 %. The matrix effect of the method was evaluated, indicating significant suppression values for the liver and the kidney, which varied respectively from -45 to -15.5 % and -35 to - 2.5%. In the same way, the other two matrices hair and bile show smaller matrix effects than the others. Finally, this method has been successfully applied to detect anabolic hormones in nighty one samples (muscle, liver, kidney, bile) available from different local butchers. Progesterone was found in 41 samples at 0.11 to 11.7 µg.kg−1 otherwise testosterone was observed in 43 samples ranging from 0.5 to 9.72 µg.kg−1

European Joint Doctorates: myth or reality?

Today, there is a lack of consensus for the full implementation of common programmes recognizing the “highest” level of higher education in Europe. Even though cotuttelle agreements are widely used for international joint supervision of PhD theses, these are merely bilateral and individual case-based agreements, far away from a real joint degree under a legal framework that establishes the programme. This article aims to describe the experience of the authors in the management and coordination of a joint doctoral programme between 2015 and 2019 and the results obtained from the interrogation of official websites about the reality in Europe concerning such programmes. Our conclusion is that, still in the 21st century, there is a huge gap to be overcome before the existence of Joint International/European Doctorates can be considered an everyday reality. Although various attempts have been made in the last 20 years, there is still a long way to go for Higher Education institutions to integrate all aspects of such programmes, and to make them something more and different than an additional Diploma Supplement. In the authors´ opinion, major efforts must be made by the administrative bodies, although the drive of the academic staff is crucial for success.  [ES] Today, there is a lack of consensus for the full implementation of common programmes recognizing the “highest” level of higher education in Europe. Even though cotuttelle agreements are widely used for international joint supervision of PhD theses, these are merely bilateral and individual case-based agreements, far away from a real joint degree under a legal framework that establishes the programme. This article aims to describe the experience of the authors in the management and coordination of a joint doctoral programme between 2015 and 2019 and the results obtained from the interrogation of official websites about the reality in Europe concerning such programmes. Our conclusion is that, still in the 21st century, there is a huge gap to be overcome before the existence of Joint International/European Doctorates can be considered an everyday reality. Although various attempts have been made in the last 20 years, there is still a long way to go for Higher Education institutions to integrate all aspects of such programmes, and to make them something more and different than an additional Diploma Supplement. In the authors´ opinion, major efforts must be made by the administrative bodies, although the drive of the academic staff is crucial for success.Coy Fuster, P.; Canovas, S.; Van Soom, A.; Bernabo, N.; Lonergan, P.; Schellander, K. (2020). European Joint Doctorates: myth or reality?. En 6th International Conference on Higher Education Advances (HEAd'20). Editorial Universitat Politècnica de València. (30-05-2020):1109-1117. https://doi.org/10.4995/HEAd20.2020.11209OCS1109111730-05-202

P-030 ACE2 receptor and its isoform short-ACE2 are expressed on human spermatozoa

STUDY QUESTION: Do human spermatozoa express angiotensin-converting enzyme 2 (ACE2) receptor? What would be its localization? SUMMARY ANSWER: Human spermatozoa express uniformly ACE2 on the sperm head and the flagellum. Moreover, the short-ACE2 isoform is concentrated on the post-acrosomal region and midpiece. WHAT IS KNOWN ALREADY: The Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) infection is generating important concerns regarding not only the possible consequences on the respiratory system, but also on other organs, including the reproductive system. ACE2 is considered the main point of entry for the SARS-CoV-2 within the cells through the binding with the spike protein on the virus surface. Furthermore, ACE2 is expressed in human testes cells including Leydig cells, Sertoli cells and spermatogonia. However, to date, the expression and location of ACE2 in mature human spermatozoa has not been investigated yet. STUDY DESIGN, SIZE, DURATION: This was an in vitro study for the evaluation of the expression and immune-localization of full-length ACE2 and its isoform, short-ACE2, in human spermatozoa. Thirthyfour non-immunized healthy normozoospermic volunteers were enrolled in the study. The study was conducted from May to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples were collected by masturbation from non-immunized healthy normozoospermic voluntaries. Motile sperm suspensions were obtained by swim-up procedure. The expression of ACE2 was assessed by Western-blot analysis, while the immune-localization of ACE2 was evaluated by immune-cytochemical analysis under confocal microscopy. Flow-cytometry experiments were also performed to assess the surface protein expression on a large number of cells. MAIN RESULTS AND THE ROLE OF CHANCE: The Western-blot analysis of sperm extracts demonstrated two specific bands, one of approximately 120 KDa, corresponding to the glycosylated full-length ACE2, and a second one of approximately 52 KDa, the molecular weight of the protein recently termed short-ACE2. The immune-cytochemical analysis showed a uniformly localization of full-length ACE2 along both the sperm head and the flagellum, whereas the short isoform was preferentially located in the post-acrosomal region of the sperm head and the midpiece. At the flow cytometer, semen samples displayed a wide between-subject variability both in the percentage of ACE2-positive spermatozoa and the density of protein surface expression. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to determine whether short-ACE2 is a cleavage product from the full-length protein or if it is originated during spermatogenesis. Moreover, the role and the interaction of ACE2 with SARS-CoV-2 in human spermatozoa should be clarified to evaluate the possible impact of the virus on sperm biology. WIDER IMPLICATIONS OF THE FINDINGS: Since mature spermatozoa are transcriptionally silent and SARS-CoV-2 is an RNA virus, it is unlikely that the virus could affect sperm biology by replicating itself. Nevertheless, the potential effects related to modifications of the sperm membrane or interaction with other receptors or specific proteins cannot be ruled out. TRIAL REGISTRATION NUMBER: not applicabl

P-440 Impact of electrospun scaffold topology on the performance of in-vitro Folliculogenesis applied to preantral ovine follicles

Study question How to improve in-vitro Folliculogenesis (ivF) protocols to address the enlarged demand of fertility preservation? Summary answer Tissue engineering-based approach opens new frontiers for ivF improving 3D-technologies addressed to support immature-ovarian-follicle-growth to obtain an increased number of competent oocytes enrolled in Assisted-Reproductive-Technology. What is known already ivF is a promising Assisted-Reproductive-Technology (ART) for preserving and restoring fertility. This technology potentially reproduces the early stages of folliculogenesis and oogenesis in-vitro allowing to move a large amount of oocyte on individual basis towards the validated protocol of in-vitro maturation/in-vitro fertilization (IVM/IVF). The current availability of biocompatible-supporting materials offers the challenging opportunity to mimic the native organ stroma in order to better reproduce the 3D environmental conditions leading to synergic follicles-oocyte development in-vitro with the aim to improve the performance of ivF in translational large sized mammal models. Study design, size, duration The present research aimed to compare preantral (PA) follicles culture on two different typologies of scaffolds fabricated using PCL(poly(epsilon caprolactone)), respectively made with patterned and randomly aligned fibers (PCL-Patterned/PCL-Randomic) with a standardized-single-follicle scaffold-free-method (3D-oil), widely validated on ovine model (Cecconi et al., 2004). The culture outcomes are compared analyzing follicle/oocyte growth, percentage of antrum differentiation and the incidence of meiotic competence, by exposing ivF growing oocytes to IVM protocol. Participants/materials, setting, methods PA follicles (mean size diameter: 250±4μm), mechanically isolated from slaughterhoused lamb ovaries, were individually cultured on electrospun PCL scaffolds (patterned vs randomic) or using the 3D-oil method. ivF were cultured alphaMEM-Fetal Bovine Serum free medium (5% Knockout Serum Replacement) supplemented with 4 IU/mL of equine Chorionic Gonadotropin (Di Berardino et al., 2021). At the end of ivF (14-days) the fully-grown oocytes isolated from early-antral follicles were tested on IVM. Main results and the role of chance PCL-Patterned electrospun scaffolds were able to strongly support a synergic oocyte and follicular growth. The 3D culture on Patterned electrospun scaffold supported the highest viability of follicles (87.5% vs 63% under 3D-oil conditions). On the contrary, the highest incidence of degenerated follicles was observed in cultures performed using PCL-Randomic materials (55 vs 37% vs 12.5% for PCL-Randomic vs 3D-oil vs PCL-Patterned, respectively; p <0.0004). The greatest follicle diameter increment (74.7±1 vs 70±0.4 vs 60.9±2%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, respectively p <0.0007) and rate of antrum differentiation (87.5% vs 45% and vs 63%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, for both p <0.0001) were observed in PA ovine follicles cultured on PCL-Patterned scaffolds. Furthermore, PCL-Patterned electrospun scaffolds supported a complete functional development of the oocyte compartment. More in detail, the majority of fully grown oocytes isolated from early- antral follicles grown on PCL-Patterned materials reached the metaphase-II stage (MII 80%) at the end of IVM in comparison to the significant lower percentage in 3D-oil (MII 68%, p =0.04) and PCL-Randomic (MII 18%, p <0.0001) protocols, respectively. Limitations, reasons for caution - Wider implications of the findings Tissue engineering scaffold-based approach represents a valid strategy generating a multi-organ in-vitro system, where different compartments may cooperate generating the complexity of paracrine-mechanism controlling early-follicles outcomes. Scaffold topology is essential to control early-follicles development. Indeed, exclusively PCL-Patterned can preserve long-term follicle 3D-microarchitecture supporting in-vitro oogenesis up to a complete meiotic-competence-acquisition. Trial registration number not applicabl

When Electrospun Fiber Support Matters: In Vitro Ovine Long-Term Folliculogenesis on Poly (Epsilon Caprolactone) (PCL)-Patterned Fibers

Current assisted reproduction technologies (ART) are insufficient to cover the slice of the population needing to restore fertility, as well as to amplify the reproductive performance of domestic animals or endangered species. The design of dedicated reproductive scaffolds has opened the possibility to better recapitulate the reproductive 3D ovarian environment, thus potentially innovating in vitro folliculogenesis (ivF) techniques. To this aim, the present research has been designed to compare ovine preantral follicles in vitro culture on poly(epsilon-caprolactone) (PCL)-based electrospun scaffolds designed with different topology (Random vs. Patterned fibers) with a previously validated system. The ivF performances were assessed after 14 days under 3D-oil, Two-Step (7 days in 3D-oil and on scaffold), or One-Step PCL protocols (14 days on PCL-scaffold) by assessing morphological and functional outcomes. The results show that Two- and One-Step PCL ivF protocols, when performed on patterned scaffolds, were both able to support follicle growth, antrum formation, and the upregulation of follicle marker genes leading to a greater oocyte meiotic competence than in the 3D-oil system. In conclusion, the One-Step approach could be proposed as a practical and valid strategy to support a synergic follicle-oocyte in vitro development, providing an innovative tool to enhance the availability of matured gametes on an individual basis for ART purposes

Pre-Treatment of Swine Oviductal Epithelial Cells with Progesterone Increases the Sperm Fertilizing Ability in an IVF Model

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional modification, called capacitation, in order to acquire their fertilizing ability. Since oviductal epithelial cells (SOECs) and progesterone (P4) are two major modulators of capacitation, here we investigated their impact on sperm functionality by using an IVF swine model. To that, we treated SOECs with P4 at 10, 100, and 1000 ng/mL before the coincubation with spermatozoa, thus finding that P4 at 100 ng/mL does not interfere with the cytoskeleton dynamics nor the cells' doubling time, but it promotes the sperm capacitation by increasing the number of spermatozoa per polyspermic oocyte (p < 0.05). Moreover, we found that SOECs pre-treatment with P4 100 ng/mL is able to promote an increase in the sperm fertilizing ability, without needing the hormone addition at the time of fertilization. Our results are probably due to the downregulation in the expression of OVGP1, SPP1 and DMBT1 genes, confirming an increase in the dynamism of our system compared to the classic IVF protocols. The results obtained are intended to contribute to the development of more physiological and efficient IVF systems