Avances en la producción in vitro e in vivo de embriones porcinos

Los objetivos generales de la presente tesis doctoral fueron incrementar la eficiencia de los sistemas actuales de PIV de embriones y la mejora de las técnicas de transferencia de embriones, y estos se concretaron en los siguientes objetivos específicos: (1) analizar la eficacia de dos combinaciones de CPAs: EG+DMSO y EG+PG para la vitrificación de ovocitos sobre los parámetros de viabilidad, fecundación y desarrollo embrionario, (2) evaluar el efecto de tres IRMs: dbcAMP, cicloheximida y cilostamida y sus interacciones con las gonadotropinas sobre la maduración meiótica y el desarrollo embrionario de ovocitos porcinos, (3) determinar los efectos de la adición de AsA, en los medios de maduración, fecundación y cultivo embrionario sobre los parámetros de maduración, fecundación y desarrollo embrionario, así como el efecto de la adición de AsA en los medios de vitrificación sobre la viabilidad después del calentamiento, (4) investigar los efectos del número de partos, la estación del año y el intervalo destete-celo sobre los parámetros embrionarios en día 6 después de la inseminación de donantes superovuladas en el celo post-destete, (5) explorar la posibilidad de re-vitrificar embriones porcinos obtenidos in vivo (6) evaluar la eficacia de los tanques de nitrógeno seco para mantener la viabilidad y la calidad de embriones porcinos obtenidos in vivo y vitrificados durante un periodo de 3 días y (7) investigar en cerdas receptoras como influye el número de partos en su capacidad reproductiva después de la NsDU-ET. Metodología: Para las técnicas in vitro, se utilizó un sistema de producción in vitro de embriones dónde los ovocitos fueron obtenidos de cerdas prepúberes procedentes de matadero. Durante el proceso, los parámetros de fecundación y desarrollo embrionario fueron evaluados. Para la vitrificación y calentamiento de embriones u ovocitos se utilizó el sistema SOPS. En las técnicas in vivo, después de la detección del estro y la inseminación artificial, los embriones fueron recolectados y evaluados. Las transferencias de embriones se realizaron de una manera no quirúrgica. Las conclusiones de la presente tesis doctoral fueron. 1. En ausencia de vitrificación, los efectos tóxicos de los medios de vitrificación sobre ovocitos en estadio de VG fueron mínimos y similares para ambas combinaciones de CPA. 2. Se pueden obtener blastocistos de alta calidad mediante la vitrificación de ovocitos inmaduros. Sin embargo, la tasa de blastocistos fue muy baja y el desarrollo embrionario de los ovocitos vitrificados fue menor comparado con el de los controles. 3. La interacción de las Gns con los tres inhibidores reversibles de la meiosis (dbcAMP, cicloheximida y cilostamida) aceleraron la progresión meiótica hasta el estadio de MII. La presencia de dbcAMP durante la primera fase de la maduración pudo incrementar, incluso duplicar la capacidad de los ovocitos para alcanzar el estadio de blastocisto. 4. La suplementación de medio de IVM/IVF/IVC con AsA (50 µg/mL) no mejoró los resultados de la PIV de embriones. Por el contrario, la adición de AsA a los medios de vitrificación y calentamiento aumentó la supervivencia después de la vitrificación. 5. El número de partos de la donante no afectó las tasas de gestación y de fecundación así como el número y calidad de los embriones de día 6, independientemente de la estación del año (desde otoño a primavera) o del IDC (3 o 4 días). 6. Los blastocistos porcinos derivados de embriones vitrificados y calentados podrían re-vitrificarse con buenas tasas de supervivencia. 7. El DS es un eficiente sistema para almacenar embriones vitrificados durante un periodo de tres días, sin afectar la viabilidad después del calentamiento. 8. Las tasas de gestación y fecundación así como el tamaño de camada no se vieron afectadas por el número de partos de las receptoras después de la transferencia no quirúrgica de embriones. The general objectives of this work were to enhance the efficiency of the IVP of porcine embryos and to improve the porcine ET technology. With this propose, the specific objectives contained in this thesis were: (1) To study the effectiveness of two combinations of CPAs, EG+DMSO or EG+PG, for the vitrification of GV stage porcine oocytes and their effects on the oocyte viability, fertilization ability, and the subsequent developmental competence (2) To evaluate the effect of three MINs (dbcAMP, cycloheximide and cilostamide) and their interactions with Gns on the meiotic maturation and developmental competence of porcine oocytes (3) To determine the effects of AsA supplementation to the IVM, IVF and IVC media, on the maturation, fertilization and embryonic developmental parameters, and to assess the effects of adding AsA to vitrification and warming defined media on the vitrification survival of IVP-porcine blastocysts (4) To investigate the effects of parity, season and WEI on the reproductive and embryonic parameters at day 6 after insemination of donor sows superovulated at postweaning estrus (5) To explore the possibility of re-vitrify in vivo-derived porcine embryos (6) To assess if the DSs is adequate for maintaining the viability and quality of vitrified in vivo-derived porcine embryos for a 3-day storage period (7) To investigate the effects of the recipients’ parity on their reproductive performance after NsDU-ET. Methodology: For the in vitro system, production of porcine embryos was performed using ovaries from prepubertal gilts at a local slaughterhouse. Fertilization and embryo development parameters were assessment. Vitrification and warming of oocytes and embryos was performed by SOPS system. For in vivo techniques, after estrus detection and artificial insemination, embryos were recovered and evaluated. Embryos were transferred by non-surgical deep uterine embryo transfer. The conclusions of this thesis were: 1. In the absence of vitrification, the toxic effects of the vitrification media on the GV oocytes were minimal and similar for both CPA combinations. 2. High-quality blastocysts can be produced from SOPS vitrified immature oocytes. However, the blastocyst rate remained very low, and the developmental competence of the vitrified oocytes was reduced compared with the non-vitrified controls. 3. The interaction of Gns with the three MINs tested (dbcAMP, cycloheximide and cilostamide) accelerated meiotic progression to the MII stage. The presence of dbcAMP during the first period maturation increased or even doubled the capacity for oocyte development to the blastocyst stage. 4. Under our experimental conditions, the supplementation of IVM/IVF/IVC media with AsA (50 μg/mL) failed to improve the IVP-outcomes. In contrast, the addition of AsA to chemically defined vitrification and warming media enhanced the blastocysts survival. 5. The parity of donor multiparous sows did not affect the pregnancy and fertilization rates and the number and quality of 6-day-old embryos, regardless of the time of the year (from fall to spring) or the WEI (3 or 4 days). 6. 6. Porcine blastocysts derived from vitrified and warmed embryos could be re-vitrified with quite good survival rates. 7. The DS is an efficient system for the storage of vitrified embryos for a storage period of three days, without affecting their viability after warming. 8. The pregnancy and farrowing rates and litter sizes were not affected by the recipients’ parity after NsDU-ET

Eventual re-vitrification or storage in liquid nitrogen vapor does not jeopardize the practical handling and transport of vitrified pig embryos

This study aimed (1) to evaluate the in vitro post-warming survival of porcine embryos after re vitrification and (2) to assess the efficacy of transport of embryos in dry shipper (DS) in maintaining the viability and quality of vitrified embryos for a 3-day period. Embryos at the compacted or cavitating morula (CCM) and unhatched blastocyst (UBL) stages were surgically obtained from weaned, crossbred sows. In the first experiment, more than 85% of the embryos survived an initial vitrification and warming and achieved comparable survival rates to those of their fresh counterparts. In contrast, those embryos subjected to a second vitrification and warming had clearly lower survival rates (60% and 64% for re vitrified embryos from the CCM and UBL groups, respectively) compared to the survival rates of the initial vitrification and fresh control groups (P amp;lt; 0.01). Hatching rates were similar in re-vitrified blastocysts derived from vitrified CCMs and fresh control groups (50.8% and 55.3%, respectively). However, differences (P amp;lt; 0.01) in hatching rates were recorded in re-vitrified blastocysts derived from vitrified UBLs and fresh control blastocysts (14.7% and 90.0%, respectively). In the second experiment, vitrified embryos were stored in a liquid nitrogen tank for one month. Then, the straws containing the embryos were transferred to a DS (DS group) or to another liquid nitrogen tank (control group) for an additional three days. Embryos from the DS and control groups had similar survival and hatching rates, regardless of the embryonic stage considered. The DS storage of CCMs and UBLs did not affect their development after culturing, including total cell numbers, compared to the control, although their apoptotic index was slightly higher (P amp;lt; 0.05), regardless of the developmental stage. In conclusion, although re-vitrification negatively affects embryo survival, this study demonstrated that amp;gt;60% of vitrified embryos could be successfully re-vitrified and re-warmed. The present study also showed the effectiveness of the DS for the storage of vitrified porcine CCMs and UBLs for at least three 3 days. (C) 2018 Elsevier Inc. All rights reserved.Funding Agencies|Ministry of Economy and Competitiveness (Madrid, Spain)/the European Regional Development Fund [AGL2015-69735-R]; Seneca Foundation, Murcia, Spain [19892/GERM/15]</p

Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 degrees C

Vitrification is the ideal method for long-lasting storage of porcine embryos. However, both strict airline regulations for transport of liquid nitrogen dewars and the technical problems experienced when vitrified embryos are transferred using non-surgical procedures have led to the introduction of alternative storage methods, such as preserving embryos in liquid state. This study evaluated whether a pH-stable medium containing high concentrations of either foetal calf serum (FCS; 50%) or BSA (4%) combined with storage at temperatures of 17 degrees C or 20 degrees C maintained in vivo-derived morulae and blastocysts alive and unhatched (a sanitary requirement for embryo transportation) during 72 h of storage. Neither FCS nor BSA supplements were able to counteract the negative effect of low temperatures (17 degrees C) on embryonic survival after storage. At 20 degrees C, the protective effect of FCS or BSA depended on embryo stage. While FCS successfully arrested embryo development of only blastocysts, BSA arrested the development of both morulae and blastocysts. Over 80% of BSA arrested embryos restarted development by conventional culture and progressed to further embryonic stages, including hatching. In conclusion, porcine morulae and blastocysts can survive and remain unhatched during at least 72 h when stored at 20 degrees C in a BSA-containing medium.Funding Agencies|Ministry of Economy and Competitiveness (Madrid, Spain)/the European Regional Development Fund [AGL2015-69735-R]; Seneca Foundation, Murcia, Spain [19892/GERM/15]; Research Council FORMAS, Stockholm [2017-00946]; Ministry of Economy and Competitiveness (Madrid, Spain) [BES2013-064087, BES-2013-064069, BES-2016-077869]</p

The overlaying oil type influences in vitro embryo production: differences in composition and compound transfer into incubation medium between oils

The oil overlay micro-drop system is widely used for cultures of mammalian gametes and embryos. We evaluated hereby the effects of two unaltered commercial oils-Sigma mineral oil (S-MO) and Nidoil paraffin oil (N-PO)-on in vitro embryo production (IVP) outcomes using a pig model. The results showed that while either oil apparently did not affect oocyte maturation and fertilization rates, S-MO negatively affected embryo cleavage rates, blastocyst formation rates, and, consequently, total blastocyst efficiency of the system. No differences in the oxidation state were found between the oils or culture media incubated under S-MO or N-PO. Although both oils slightly differed in elemental composition, there were no differences in the concentrations of elements between fresh media and media incubated under oils. By contrast, we demonstrated clear oil-type differences in both the composition of volatile organic compounds (VOC) and the transfer of some of these VOCs (straight-chain alkanes and pentanal and 1,3-diethyl benzene) to the culture medium, which could have influenced embryonic development.Funding Agencies|MINECO-FEDER, Madrid, Spain [AGL2015-69735-R]; Fundacion Seneca, Murcia, Spain [19892/GERM/15]; MINECO [BES-2013-064087, BES-2013-064069]</p

Interspecies Chimerism with Mammalian Pluripotent Stem Cells

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos