Nuevas estrategias para mejorar la eficiencia del semen sexado en producción in vitro de embriones bovinos

Tesis para obtener el grado de Doctor en Ciencias Veterinarias, de la Universidad Nacional del Litoral, en 2017El objetivo de esta tesis fue el de optimizar el uso de semen sexado en embriones bovinos producidos in vitro (PIVE). Para ello se realizaron tres estudios con el fin de evaluar: diferentes medios de fecundación in vitro (FIV): M199-FERT, fluido oviductal sintético (SOF-FERT) y Tirodes-albúmina-lactato-piruvato (TALP) (Estudio 1); agentes hiperactivadores de la motilidad espermática y tiempos de fecundación (Estudio 2) y mini-coloides de densidad variable (Estudio 3). En el Estudio 1, complejos ovocito-cumulus (COCs) fueron fecundados con semen sexado. Se analizó el clivaje, polispermia, formación de blastocitos, calidad embrionaria y capacidad de eclosionar. Los resultados mostraron que el medio de fecundación afectó la tasa de clivaje, desarrollo embrionario, calidad y capacidad de eclosión. En el Estudio 2, factores hiperactivadores de la motilidad: cafeína (CA), teofilina (TH) y CA + TH fueron testados al igual que la duración de la fecundación (8 hs versus 18 hs). Los factores hiperactivadores de la motilidad afectaron las tasas de clivaje y desarrollo embrionario, mientras que el acortamiento de la fecundación no afectó la producción embrionaria. Finalmente, en el Estudio 3 se analizaron diferentes mini-coloides de densidad continuos y discontinuos. La tasa de recuperación y viabilidad espermática y la calidad seminal se vio afectada por el tipo de mini-coloides de densidad. La mono-capa de densidad continua al 80% y el medio SOF-FERT combinado con CA + TH resultaron ser la mejor combinación para obtener la mayor tasa de recuperación, viabilidad y calidad espermática, tasa de clivaje, desarrollo embrionario, calidad embrionaria y capacidad de eclosión.EEA RafaelaFil: Ferre, Luis Bernardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina

Efecto de la suplementación con ciclodextrinas cargadas con colesterol al semen sexado bovino post-descongelación para su uso en producción in vitro de embriones

Los espermatozoides bovinos presentan una bajo relación de colesterol/fosfolípidos en la membrana plasmática. Esta característica es importante ya que el colesterol juega un papel fundamental en el mantenimiento de la estructura y funcionalidad de la membrana espermática tras la congelación, y, por lo tanto, en la supervivencia del espermatozoide tras el proceso de criopreservación. El proceso de sexado podría agravar todavía aún más esta situación. El objetivo de este trabajo es estabilizar las membranas plasmáticas de espermatozoides sexados mediante el agregado de ciclodextrinas cargadas con colesterol (CLC) post-descongelado y consecuentemente, prolongar su funcionalidad, aumentar su capacidad de fecundar ovocitos in vitro y producción de embriones. Para ello se trataron espermatozoides sexados con CLC a diferentes concentraciones e incubados por 15 minutos antes de evaluar el patrón de motilidad mediante un sistema computarizado CASA y fertilizar ovocitos in vitro. Los espermatozoides tratados con 3 mg de metil-β-ciclodextrina saturada de colesterol cada 120x106 espermatozoides presentaron mejor motilidad y vigor y se mantuvieron elevados durante el tiempo analizado. La concentración de 3 mg de CLC también produjo mayor clivaje, MOR/eBL, BL y embriones totales.Bovine sperm have a low cholesterol/phospholipids ratio in the sperm plasma membrane. This characteristic is important due to cholesterol plays a key role in maintaining the structure and function of sperm membrane after freezing, and therefore, increasing sperm survival. This situation could be amplified in case of sexed semen. The aim of this study is to stabilize the plasma membranes of sperm sexed by adding cholesterol-loaded cyclodextrins (CLC) post-thaw and consequently extend their functionality, increase their ability to fertilize oocytes and produce embryos in vitro. Sexed sperm were treated with CLC at different concentrations and incubated for 15 minutes before evaluating different motility parameters by a computerized system (CASA) and fertilize oocytes in vitro. Sperm treated with 3 mg of methyl-β-cyclodextrin saturated cholesterol for each 120x106 of sperm, showed higher motility parameters and remained high during the analyzed period. The concentration of 3 mg of CLC also increased cleavage rate, early (MOR/eBL), late (BL) and total embryo development.EEA RafaelaFil: Ferre, Luis Bernardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina.Fil: Grötter, L. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Fresno, C. National Institute of Genomic Medicine (INMEGEN). Computational Genomics Division; MéxicoFil: Cattáneo, Luciano. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentin

7 & 7 Synch and 7-day progesterone- based protocols for estrus synchronization prior fixedtime artificial insemination in multiparous beef sucked cows

PosterEstrogen-based protocols combined with a progestinreleasing intravaginal device (P4RID) and prostaglandin (PG) synchronize follicle wave emergence prior to artificial insemination (AI) in Bos taurus, with pregnancies per AI (P/AI) ranging from 40 to 60%. Gonadotropin-releasing hormone (GnRH) based protocols + P4RID + PG rely on the presence of a physiologically mature dominant follicle at initial GnRH. Failure to induce ovulation represents a major obstacle to improve P/AI. Pre-synchronization by PG + P4RID 7 days in advance of GnRH increase likelihood of ovulation following GnRH. The aim of this trial was to evaluate fertility in suckled beef cows using GnRH or estrogen treatments with or without pre-synchronizationEEA BarrowFil: Ferré, Luis Bernardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Chacra Experimental Integrada Barrow; Argentina.Fil: Jaeschke, J. Biogénesis Bagó; Argentina.Fil: Gatti, J. Biogénesis Bagó; Argentina.Fil: Baladón, G. Biogénesis Bagó; Argentina.Fil: Bellocq, E. Biogénesis Bagó; Argentina.Fil: Fernández, G. Actividad privada; Argentina.Fil: Thomas, J. University of Missouri. Division of Animal Sciences; Estados Unido

Recent progress in bovine In vitro‐derived embryo cryotolerance: impact of In vitro culture systems, advances in cryopreservation and future considerations

Cryopreservation of in vitro‐derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra‐low temperature storage method in order to maximize donor ovum pick‐up (OPU) turn‐over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled‐rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in‐straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.EEA BarrowFil: Ferre, Luis Bernardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Barrow; Argentina.Fil: Kjelland, M.E. Conservation, Genetics and Biotech, LLC; Estados Unidos. Mayville State University; Estados UnidosFil: Taiyeb, A.M. Conservation, Genetics and Biotech, LLC; Estados Unidos. Barz IVF Center for Embryo Research and Infertility Treatment; IrakFil: Campos‐Chillon, L.F. California Polytechnic State University. Animal Science Department; Estados UnidosFil: Ross, Pablo J. University of California Davis. Department of Animal Science; Estados Unido

Llama (Lama glama) semen collection via thermo-electric artificial vagina: effect of seasonality and collection interval on ejaculate characteristics

Adult male Llamas (Lama glama) (n = 6) were collected using a receptive female and a thermo-electric artificial vagina assembled with a polyethylene collection bag with the following objectives: (a) to develop a reliable and repeatable semen collection technique for Llama, (b) validate semen evaluation tests for Llama, and (c) to determine the effect of collection frequency and season on Llama semen characteristics. First, the semen and sperm variables were recorded and validated through their own repeatability. Semen collection intervals tested were: 1X/week for three weeks and 3X/week for another three weeks, the second collection period occurring after two weeks of sexual rest. The collection frequency of 3X/week significantly reduced (P < 0.05) the sperm viability, motility, concentration, Total Sperm/Ejaculate, Total Motile Sperm/Ejaculate and Total Live Sperm/Ejaculate, but improved Total Sperm/Week and Total Live Sperm/Week. All recorded variables were significantly higher during the summer in comparison to the spring with the exception of morphology abnormalities, volume, and viability. Also, the 1X/week versus the 3X/week semen collection frequencies did not produce a significant difference in the percent of total motile sperm/week. Based on semen and sperm characteristics evaluated herein, Llama semen collected in the summer was better (P < 0.05), with regard to the majority of the variables analyzed, than semen collected in the spring.EEA BalcarceFil: Ferré Luis Bernardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Malik, Genoveva. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Alberio, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Fresno, Cristóbal. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Católica de Córdoba. Facultad de Ingeniería; Argentina.Fil: Kjelland, Michael. Conservation, Genetics & Biotech; Estados Unidos

Transvaginal ultrasound-guided oocyte retrieval in cattle: state-of-the-art and its impact on the in vitro fertilization embryo production outcome

Transvaginal ultrasound-guided oocyte retrieval (commonly called OPU) and in vitro embryo production (IVP) in cattle has shown significant progress in recent years, in part, as a result of a better understanding of the full potential of these tools by end users. The combination of OPU and IVP (OPU-IVP) has been successfully and widely commercially used worldwide. The main advantages are a greater number of embryos and pregnancies per unit of time, faster genetic progress due to donor quick turn around and more elite sires mating combinations, larger spectrum of female age (calves, prepuberal, heifer, cow) and condition (open, pregnant) from which to retrieve oocytes, a reduced number of sperm (even sexed) required to fertilize the oocytes, among other benefits. OPU-IVP requires significant less donor preparation in comparison to conventional embryo transfer (<50% of usual FSH injections needed) to the extent of no stimulating hormones (FSH) are necessary. Donor synchronization, stimulation, OPU technique, oocyte competence, embryo performance, and its impact on cryopreservation and pregnancy are discussed.EEA BarrowFil: Ferre, Luis Bernardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Barrow; Argentina.Fil: Alvarez-Gallardo, Horacio. Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán. Laboratorio de Reproducción; MéxicoFil: Romo, Salvador. Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán. Laboratorio de Reproducción; MéxicoFil: Fresno, Cristóbal. Anáhuac University of México. Health Sciences Research Center (CICSA); MéxicoFil: Stroud, Todd. Hoofstock Genetics; Estados UnidosFil: Stroud, Brad. Stroud Veterinary Embryo Services, Inc.; Estados UnidosFil: Lindsey, Brad. Ovitra Biotechnology, Inc.; Estados UnidosFil: Kjelland, M.E. Conservation, Genetics and Biotech, LLC; Estados UnidosFil: Kjelland, M.E. Mayville State University; Estados Unido

Differences in mortality in critically ill elderly patients during the second COVID-19 surge in Europe

Background!#!The primary aim of this study was to assess the outcome of elderly intensive care unit (ICU) patients treated during the spring and autumn COVID-19 surges in Europe.!##!Methods!#!This was a prospective European observational study (the COVIP study) in ICU patients aged 70 years and older admitted with COVID-19 disease from March to December 2020 to 159 ICUs in 14 European countries. An electronic database was used to register a number of parameters including: SOFA score, Clinical Frailty Scale, co-morbidities, usual ICU procedures and survival at 90 days. The study was registered at ClinicalTrials.gov (NCT04321265).!##!Results!#!In total, 2625 patients were included, 1327 from the first and 1298 from the second surge. Median age was 74 and 75 years in surge 1 and 2, respectively. SOFA score was higher in the first surge (median 6 versus 5, p &amp;lt; 0.0001). The PaO!##!Conclusion!#!An unexpected, but significant, decrease in 30-day and 90-day survival was observed during the second surge in our cohort of elderly ICU patients. The reason for this is unclear. Our main concern is whether the widespread changes in practice and treatment of COVID-19 between the two surges have contributed to this increased mortality in elderly patients. Further studies are urgently warranted to provide more evidence for current practice in elderly patients.!##!Trial registration number!#!NCT04321265 , registered March 19th, 2020

Health-related quality of life in older patients surviving ICU treatment for COVID-19: results from an international observational study of patients older than 70 years

International audienceBackground health-related quality of life (HRQoL) is an important patient-centred outcome in patients surviving ICU admission for COVID-19. It is currently not clear which domains of the HRQoL are most affected. Objective to quantify HRQoL in order to identify areas of interventions. Design prospective observation study. Setting admissions to European ICUs between March 2020 and February 2021. Subjects patients aged 70 years or older admitted with COVID-19 disease. Methods collected determinants include SOFA-score, Clinical Frailty Scale (CFS), number and timing of ICU procedures and limitation of care, Katz Activities of Daily Living (ADL) dependence score. HRQoL was assessed at 3 months after ICU admission with the Euro-QoL-5D-5L questionnaire. An outcome of ≥4 on any of Euro-QoL-5D-5L domains was considered unfavourable. Results in total 3,140 patients from 14 European countries were included in this study. Three months after inclusion, 1,224 patients (39.0%) were alive and the EQ-5D-5L from was obtained. The CFS was associated with an increased odds ratio for an unfavourable HRQoL outcome after 3 months; OR 1.15 (95% confidence interval (CI): 0.71–1.87) for CFS 2 to OR 4.33 (95% CI: 1.57–11.9) for CFS ≧ 7. The Katz ADL was not statistically significantly associated with HRQoL after 3 months. Conclusions in critically ill old intensive care patients suffering from COVID-19, the CFS is associated with the subjectively perceived quality of life. The CFS on admission can be used to inform patients and relatives on the risk of an unfavourable qualitative outcome if such patients survive

The association of the Activities of Daily Living and the outcome of old intensive care patients suffering from COVID-19

Purpose: Critically ill old intensive care unit (ICU) patients suffering from Sars-CoV-2 disease (COVID-19) are at increased risk for adverse outcomes. This post hoc analysis investigates the association of the Activities of Daily Living (ADL) with the outcome in this vulnerable patient group. Methods: The COVIP study is a prospective international observational study that recruited ICU patients >= 70 years admitted with COVID-19 (NCT04321265). Several parameters including ADL (ADL; 0 = disability, 6 = no disability), Clinical Frailty Scale (CFS), SOFA score, intensive care treatment, ICU- and 3-month survival were recorded. A mixed-effects Weibull proportional hazard regression analyses for 3-month mortality adjusted for multiple confounders. Results: This pre-specified analysis included 2359 patients with a documented ADL and CFS. Most patients evidenced independence in their daily living before hospital admission (80% with ADL = 6). Patients with no frailty and no disability showed the lowest, patients with frailty (CFS >= 5) and disability (ADL < 6) the highest 3-month mortality (52 vs. 78%, p < 0.001). ADL was independently associated with 3-month mortality (ADL as a continuous variable: aHR 0.88 (95% CI 0.82-0.94, p < 0.001). Being "disable" resulted in a significant increased risk for 3-month mortality (aHR 1.53 (95% CI 1.19-1.97, p 0.001) even after adjustment for multiple confounders. Conclusion: Baseline Activities of Daily Living (ADL) on admission provides additional information for outcome prediction, although most critically ill old intensive care patients suffering from COVID-19 had no restriction in their ADL prior to ICU admission. Combining frailty and disability identifies a subgroup with particularly high mortality