Factors affecting the in vitro embryo production in cattle associated to ovum pick up sistem

La producción de embriones mediante la recuperación de ovocitos inmaduros por ovum pick up (OPU), y su posterior maduración, fecundación y cultivo en el laboratorio in vitro, presenta numerosos beneficios para optimizar el potencial reproductivo, tanto de hembras como de machos. Además, frente a la superovulación convencional mediante tratamiento hormonal y la recogida de embriones in vivo, la producción in vitro de embriones (PIVE) con ovocitos de OPU ofrece considerables ventajas. Sin embargo, actualmente la PIVE continua siendo ineficiente e incapaz de producir embriones de calidad similar a los in vivo, lo cual ha limitado una aplicación más amplia de esta tecnología. Así pues, el objetivo de esta tesis fue la optimización de la PIVE en ganado vacuno, condicionado por las peculiaridades y deficiencias de la PIVE cuando los ovocitos son recuperados por la técnica de OPU. Con este fin, cinco experimento se llevaron a cabo en esta tesis. En el primero de ellos se estudió el efecto del fluido oviductal bovino (FOb) sobre el desarrollo y la calidad embrionaria (Experimento 1). Las fases del proceso de PIVE en las cuales el cultivo de ovocitos/embriones, bien individualmente o bien en número reducido, pudiera perjudicar el posterior desarrollo hasta el estadio de blastocisto y/o a su calidad, se estudiaron en el Experimento 2. En el Experimento 3 se testó si el desarrollo y la calidad de embriones cultivados in vitro en número reducido podría ser mejorada con la adición conjunta de factor de crecimiento epidérmico, insulina, transferrina y selenio (FCE-ITS) o por el sistema de cultivo de embriones llamado well of well (WOW). Las propiedades protectoras de la melatonina frente a los daños causados por el estrés oxidativo, subsecuentes de las condiciones de PIVE o de un estrés térmico durante la maduración ovocitaria, fueron evaluadas en el Experimento 4. Por último, en el Experimento 5 usamos ovocitos recolectados por OPU para evaluar el efecto del semen sexado sobreCebrián Serrano, A. (2013). Factors affecting the in vitro embryo production in cattle associated to ovum pick up sistem [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/27646Palanci

Male pronucleus formation after ICSI: effect of oocyte cysteine or sperm Triton X-100 treatments

In pigs, intracytoplasmic sperm injection (ICSI) efficiency is still poor. The inadequate decondensation of the sperm chromatin, its transformation into the male pronucleus (MPN) together with the subsequent inability to activate the oocyte, seem to be the main causes of the low ICSI efficiency. In order to improve the MPN formation we took two different approaches. On the one hand, the in vitro culture (IVC) medium postICSI was supplemented with 1.71mM cysteine (CYS). Alternatively, the sperm membrane was digested with Triton X-100 (TX) before ICSI, to improve the exposure of the sperm chromatin to the oocyte cytoplasm. After 6 h post-ICSI, the activation rate was significantly higher in TX group (70.0%) compared with CYS and control groups (42.2% and 48.9%, respectively; P < 0.05). However, no significant differences between the three groups were observed in terms of the number of pronuclei, 2PN (oocytes with 2 pronuclei and no visible sperm), and 1PN + sperm (oocytes with 1 pronucleus and one sperm head). At 22 h post-ICSI, the activation rates were similar in TX, CYS, and control groups (73.1, 78.9, and 75.7%, respectively). In addition, we did not observe significant differences between TX, CYS, and control groups for the number of pronuclei, 2PN (52.6, 56.7, and 50%, respectively) or 1PN + sperm (21.1, 33.3, and 32.1%, respectively). While no cleavage was observed in the CYS group, no significant differences in the cleavage rate were observed between control (21.3%) and TX (10.5%) groups. In summary, and under our conditions, neither CYS supplement, nor sperm TX pre-treatment were able to improve MPN formation at 6 and 22 h post-ICSI. However, the sperm TX pre-treatment improved oocyte activation at 6 h post-ICSI, although 22 h post-ICSI such a beneficial effect did not persist

Inter-Rater Variability in the Evaluation of Lung Ultrasound in Videos Acquired from COVID-19 Patients

12 páginas, 7 figuras, 1 tablaLung ultrasound (LUS) allows for the detection of a series of manifestations of COVID-19, such as B-lines and consolidations. The objective of this work was to study the inter-rater reliability (IRR) when detecting signs associated with COVID-19 in the LUS, as well as the performance of the test in a longitudinal or transverse orientation. Thirty-three physicians with advanced experience in LUS independently evaluated ultrasound videos previously acquired using the ULTRACOV system on 20 patients with confirmed COVID-19. For each patient, 24 videos of 3 s were acquired (using 12 positions with the probe in longitudinal and transverse orientations). The physicians had no information about the patients or other previous evaluations. The score assigned to each acquisition followed the convention applied in previous studies. A substantial IRR was found in the cases of normal LUS (κ = 0.74), with only a fair IRR for the presence of individual B-lines (κ = 0.36) and for confluent B-lines occupying 50% (κ = 0.50). No statistically significant differences between the longitudinal and transverse scans were found. The IRR for LUS of COVID-19 patients may benefit from more standardized clinical protocols.This research was partially funded by CDTI (Spanish acronym: Centre for Industrial Tech- nological Development), funding number COI-20201153. Partially supported by the Google Cloud Research Credits program with the funding number GCP19980904, by the project RTI2018-099118- A-I00 founded by MCIU/AEI/FEDER UE and by the European Commission–NextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global)

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Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Beneficial Effect of Two Culture Systems with Small Groups of Embryos on the Development and Quality of In Vitro-Produced Bovine Embryos

Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500l medium) and three smaller groups [five embryos/50l medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured

Effect of number of oocytes and embryos on in vitro oocyte maturation, fertilization and embryo development in bovine

The aim of this study was to identify the in vitro development stage at which the culture of a single or low number (n = 5 or 10) of oocytes/embryos could impair development in comparison with culture in group (n = 50). In the Experiment 1, it was confirmed that single in vitro embryo production yielded lower cleavage and blastocyst rates than in group (49.4 vs. 83.0%; 0% vs. 37.8%, respectively; p < 0.05). In Experiment 2 and 3, it was observed no effect on embryo development of culturing single or low number of oocytes during maturation and fertilization, respectively. In Experiment 4, it was observed a detrimental effect on blastocyst rate when cultured single or low number of embryos during post-fertilization in vitro culture (2.9; 10.2-10.8; 33.2% in single, low number of embryos (5-10), and controlgrouped, respectively; p < 0.05). In Experiment 5, it was observed that the last part of the culture period (day 3 onwards) seemed to be more affected by the low number of embryos placed in culture. In conclusion, post-fertilization culture, especially on days 3 to 7 after fertilization, seems to be the most important stage for embryo development on single and/or low number (5-10) of embryos culture