EFFECT OF FEED RESTRICTION IN A RABBIT LINE SELECTED FOR GROWTH RATE ON REPRODUCTIVE PERFORMANCE: OVULATION INDUCTION, OOCYTE QUALITY, EMBRYONARY AND FETAL LOSSES

Tesis por compendio[EN] The general aim of this thesis was to reproductively characterize females from a rabbit line selected for growth rate and to evaluate the effect of different nutritional strategies (ad libitum and restricted), in order to improve the reproductive performance. In chapter 1, the influence of maternal and embryonic genotype on prenatal survival and fetal growth was evaluated and contrasted with a maternal line. Prenatal survival, fetal weight and fetal placenta weight were affected by both embryonic and maternal genotype. Nevertheless, no differences were detected either at transcriptomic level in fetal placenta or in progesterone and IGF-I plasma levels in these females. It may be concluded that in rabbit females from paternal lines both embryonic and maternal genotypes are key factors in the reproductive performance of these females. The aim of chapter 2 was to explore the causes of ovulation failures in these rabbit females. Results showed that non-ovulated females presented lower LH plasma concentration as well as higher body weight and leptin and BOHB plasma levels than ovulated females. Thus, ovulation failures in females from line R could be attributed to decreased LH plasma concentrations in these females which may be related with their higher body weight and leptin levels. The following three chapters were focused on improvement of the reproductive performance of these females through a different nutritional strategy: a feed-to-appetite diet of these females after the rearing period and prior to insemination. The initial hypothesis was that the females are submitted to a restricted nutritional regimen which is not enough to cope with their needs during reproduction, causing long-term disturbances of energy balance which leads to the subsequent reproductive problems. Chapter 3 aimed to determine if a feed-to-appetite nutritional strategy would affect the hypothalamus-hypophysis axis and the quality of the produced oocytes, by transcriptomic analysis. While no differences were found in the microarray analysis of the hypothalamus-hypophysis, small differences were detected in the transcript expression analysis in oocytes of a group of genes selected. MSY2 was found to be downregulated in oocytes from restricted females. As a key regulator of maternal RNA transcription and translation, changes in this essential gene could explain some of the reproductive problems of these females. Whether the differences found at oocyte level were inherent at embryonic level and so involved in the drop of fertility was studied in Chapter 4. Although no significant differences were revealed in ovulation, embryo recovery, and implantation rate, higher fetal and gestational losses were found in restricted females, as well as lower fetal growth. Thus, we concluded that the nutritional strategy employed may have an impact on the oocyte (Chapter 3), but we also demonstrated that these changes were inherited by the embryo, and result in disturbances in gestational losses and fetal growth. The final chapter of this thesis was conducted to determine whether these effects on reproductive and metabolic elements were also evident in females following the common semi-intensive farm production system. Although the results obtained showed small variances in NEFAs and BOHB plasma levels, and also in body weight, no differences were detected in global reproductive performance in terms of fertility, prolificacy and productivity. The results obtained established that although differences are found at oocyte level and inherited by embryo and fetus, no improvements are reached with the proposed nutritional strategy in terms of reproductive performance when females selected for growth rate lead a normal semi-intensive production system.[ES] El objetivo general fue la caracterización reproductiva de las hembras de una línea de conejo seleccionada por velocidad de crecimiento y el efecto de distintas estrategias nutricionales (ad libitum y restringidas), con la finalidad de mejorar el rendimiento reproductivo. En el capítulo 1 se evaluó la influencia de los genotipos materno y embrionario en la supervivencia prenatal y crecimiento fetal, entre las hembras de esta línea y una línea maternal. La supervivencia prenatal, peso fetal y el peso de la placenta resultaron afectados por los genotipos tanto embrionario como materno, pero no se detectaron diferencias sobre la placenta fetal a nivel del transcriptoma ni en los niveles de progesterona e IGF-I. Por lo tanto, se puede concluir que en éstas tanto el genotipo embrionario como el materno son factores clave en su rendimiento reproductivo. El objetivo del capítulo 2 fue explorar las causas de los fallos reproductivos en estas hembras. Los resultados mostraron que las hembras que no ovularon presentaban menores niveles de LH, un mayor peso corporal y mayor concentración en sangre de leptinas y BOHB, que aquellas que había ovulado. Por ello, los fallos en ovulación detectados en estas hembras podrían estar relacionados con una reducción en los niveles de LH, consecuencia del mayor peso de estas hembras y de los incrementados niveles de leptinas. Los siguientes capítulos se enfocaron a la mejora del rendimiento reproductivo de estas hembras empleando una estrategia nutricional distinta: la ingesta ad libitum de alimento tras el periodo de crianza hasta el momento del comienzo de su vida reproductiva. La hipótesis de partida fue que estas hembras son sometidas a un régimen nutricional restringido que no es suficiente para satisfacer sus necesidades energéticas durante la reproducción, lo que causa alteraciones en su balance energético que se manifestarían en los problemas reproductivos observados. El capítulo 3 trató de determinar a través de un análisis trasncriptómico si la estrategia nutricional planteada afectaría el eje hipotalámico-hipofisario y la calidad de los ovocitos. Aunque no se encontraron diferencias en el análisis de un micrarray realizado sobre el hipotálamo-hipófisis, sí que se detectaron en la expresión génica de los ovocitos. El transcrito MSY2 mostró una menor expresión en los ovocitos de las hembras restringidas. Este gen es un regulador clave en la maduración ovocitaria, por lo tanto, cambios en la expresión de este gen podrían explicar algunos de los problemas reproductivos de estas hembras. En el capítulo 4 se estudió si las diferencias a nivel ovocitario eran heredadas por el embrión pudiendo causar la baja fertilidad de estas hembras. Aunque no se encontraron diferencias en las tasas de ovulación, recuperación embrionaria e implantación, sí que aparecieron diferencias en las pérdidas fetales y gestacionales, así como un menor crecimiento fetal en los embriones procedentes de hembras con restricción alimentaria. Por ello, concluimos que la estrategia nutricional empleada tiene unas consecuencias en el ovocito (Capítulo 3), y demostramos que estos cambios parecen continuar en el embrión, resultando en alteraciones en pérdidas gestacionales y crecimiento fetal. El capítulo final fue desarrollado para evidenciar si los efectos reproductivos y metabólicos observados en los capítulos previos se manifestaban en las hembras que se encuentran en un sistema de producción tradicional en granja. A pesar de que los resultados mostraron variaciones en los niveles circulantes de NEFAs y BOHB y de peso corporal, no se encontraron diferencias en el rendimiento reproductivo global a nivel de fertilidad, prolificidad y productividad. Los resultados obtenidos parecen indicar que a pesar de las diferencias encontradas a nivel ovocitario, embrionario y fetal, con el régimen nutricional propuesto no se logra alcanzar mejoras en la eficiencia reproductivo de las h[CA] L'objectiu general va ser la caracterització reproductiva de les femelles d'una línia de conill seleccionada per velocitat de creixement i l'efecte de diferents estratègies nutricionals (ad libitum i restringides), amb la finalitat de millorar el rendiment reproductiu. En el capítol 1 s'avaluà la influència dels genotipus matern i embrionari en la supervivència prenatal i el creixement fetal, entre les femelles d'aquesta línia i les de una altra línia maternal. La supervivència prenatal, el pes fetal i el pes de la placenta resultaren afectats pels genotipus embrionari i matern, però no es detectaren diferències en la placenta fetal a nivell de trascriptoma ni en els nivells de progesterona i IGF-I. Per tant, en les femelles de conill seleccionades per velocitat de creixement, tant el genotipus embrionari com el matern són factors clau en el seu rendiment reproductiu. L'objectiu del capítol 2 va ser explorar les causes de les fallades reproductives en aquestes femelles. Les femelles que no ovularen presentaren menor nivells de LH, major pes corporal i major concentració de leptines i BOHB que aquelles que sí que hi havia ovulat. Per això, les fallades d'ovulació detectades en aquestes femelles podrien estar relacionades amb la reducció en els nivells de LH, com a conseqüència del major pes d'aquestes femelles i dels incrementats nivells de leptines. Els capítols següents s'enfocaren a la millora del rendiment reproductiu d'aquestes femelles mitjançant una estratègia nutricional distinta: la ingesta ad libitum d'aliment després del període de criança i fins al moment de l'inici de la vida reproductiva. La hipòtesi de partida fiu que aquestes femelles són sotmeses a un règim nutricional restringit que no és suficient per a satisfer les seves necessitats energètiques durant la reproducció, la qual cosa provoca alteracions en el balanç energètic que podrien manifestar-se en els problemes reproductius observats. El capítol 3 tractà de determinar mitjançant un anàlisi trasncritòmic si la estratègia nutricional plantejada podria afectar l'eix hipotalàmic-hipofisiari i la qualitat dels ovòcits. Metre que no es detectaren diferències en el anàlisi del microarray realitzat en el hipotàlem-hipòfisi, sí que es detectaren en l'expressió gènica del ovòcits. El transcrit MSY2 mostrà una menor expressió en els ovòcits de les femelles restringides. Aquest gen es un regulador clau en la maduració ovocitària, per aquest motiu, canvis en la seva expressió gen podrien explicar alguns dels problemes reproductius de les femelles. En el capítol 4 s'estudià si aquestes diferències a nivell ovocitari eren heretades per l'embrió i podrien causar la baixa fertilitat d'aquestes femelles. Encara que no se trobaren diferències en les taxes d'ovulació, recuperació embrionària i implantació, sí que es trobaren diferències en les pèrdues fetals i gestacionals, així com un menor creixement fetal en els embrions de les femelles provinents d'un règim alimentari restringit. Per això concloíem que l'estratègia nutricional emprada té conseqüències en el ovòcit (Capítol 3), i demostrarem que aquest canvis pareixen continuar en l'embrió, resultant en alteracions en pèrdues gestacionals i creixement fetal. El capítol final fou desenvolupat per evidenciar si els efectes reproductius i metabòlics observats en els capítols previs eren manifestats en les femelles que es troben en un sistema productiu tradicional de granja. Tot i que el resultats mostraren variacions en els nivells circulants de NEFAs i BOHB i pes corporal, no es trobaren diferències en el rendiment reproductiu global, en termes de fertilitat, prolificitat i productivitat. Els resultats obtinguts pareixen indicar que tot i que les diferències trobades a nivell ovocitari i embrionari amb un efecte significatiu en el desenvolupament i creixement fetal, amb el règim nutricional proposat no s'aconsegueix assolir millores enNaturil Alfonso, C. (2016). EFFECT OF FEED RESTRICTION IN A RABBIT LINE SELECTED FOR GROWTH RATE ON REPRODUCTIVE PERFORMANCE: OVULATION INDUCTION, OOCYTE QUALITY, EMBRYONARY AND FETAL LOSSES [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/73065TESISCompendi

Procrastination: the poor time management among university students

[EN] Academic procrastination is a fact related to the delay or postpone of academic work until last minute. This phenomenon is evident in a vast majority of university students, and its occurrence is increasing. In order to analyse possible causes and/or solutions, we studied if longer time for accomplishing an assignment incentives or avoids procrastination among university students. Results showed that both short and long time-frame groups tended to procrastinate in the same way. Additionally, academic grades did not revealed differences between groups, as the procrastination was the same between groups. Thus, this study shows that even with longer period of time to accomplish a task, university students tend to procrastinate, and thus seem to have a negative effect on their assignment grades. Therefore, it seems a current problem and measures should be developed in order to solve it.This project has received funding from the Vicerectorado de Estudios, Calidad y Acreditación of the Universitat Politècnica de València (UPV) under Proyectos de Innovación y Mejora Educativa programme (PIME/2017/B/010) and the School of Agricultural Engineering and Environment (ETSIAMN) of the Universitat Politècnica de València (UPV).http://ocs.editorial.upv.es/index.php/HEAD/HEAD18Naturil-Alfonso, C.; Peñaranda, D.; Vicente, J.; Marco-Jiménez, F. (2018). Procrastination: the poor time management among university students. Editorial Universitat Politècnica de València. 1-8. https://doi.org/10.4995/HEAD18.2018.8167OCS111518115

Feed restriction regime in a rabbit line selected for growth rate alters oocyte maturation manifested by alteration in msy2 gene expression

[EN] Young rabbit females selected for growth rate may have nutritional needs, which may not be met with the common practice of feed restriction during rearing in commercial rabbit production. The aim of this study was to analyse whether two different feeding programmes: ad libitum or restricted (130 g/day) feeding, applied in young rabbit females for 1 month at the end of rearing, could modulate the origin of ovulation process and the quality of the oocytes. At 16 weeks of age, 34 females were randomly assigned to restricted or ad libitum feeding, maintaining these conditions for a month. Then, in an initial experiment, transcriptional profiling of hypothalamus-hypophysis tissue was performed to assess failure to ovulate. In the second experiment, the gene expression analysis of some candidate genes related to oocytes quality was performed. Our results demonstrated that neither of the two feeding programmes modified the transcription of hypothalamus-hypophysis tissue, while the only differences in MSYR expression were found in in vivo mature oocytes ready for successful fertilization. Specifically, MSYR was over-expressed in oocytes from females fed ad libitum. MSYR is one of the most abundant proteins in the oocyte and has proven to be a key regulator of maternal RNA transcription and translation. This finding suggests that MSYR gene is a promising gene in our understanding of the relationship between high growth rate and reproductive performance decline.This work was supported by the Spanish Research Projects AGL2014-53405-C2-P and AGL2011-30170-C02-01 (CICYT). Carmen Naturil was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2013/296).Naturil Alfonso, C.; Peñaranda, D.; Vicente Antón, JS.; Marco-Jiménez, F. (2017). Feed restriction regime in a rabbit line selected for growth rate alters oocyte maturation manifested by alteration in msy2 gene expression. Reproduction in Domestic Animals. 52(6):976-984. https://doi.org/10.1111/rda.13006S976984526Alexander, B. M., Kiyma, Z., McFarland, M., Van Kirk, E. A., Hallford, D. M., Hawkins, D. E., … Moss, G. E. (2007). Influence of short-term fasting during the luteal phase of the estrous cycle on ovarian follicular development during the ensuing proestrus of ewes. Animal Reproduction Science, 97(3-4), 356-363. doi:10.1016/j.anireprosci.2006.01.012Armstrong, D. G., McEvoy, T. G., Baxter, G., Robinson, J. J., Hogg, C. O., Woad, K. J., … Sinclair, K. D. (2001). Effect of Dietary Energy and Protein on Bovine Follicular Dynamics and Embryo Production In Vitro: Associations with the Ovarian Insulin-Like Growth Factor System1. Biology of Reproduction, 64(6), 1624-1632. doi:10.1095/biolreprod64.6.1624Ashworth, C. J., Beattie, L., & Antipatis, C. (1999). 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Expression of MSY2 in Mouse Oocytes and Preimplantation Embryos1. Biology of Reproduction, 65(4), 1260-1270. doi:10.1095/biolreprod65.4.126

Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification

[EN] Parthenote embryos offer multiple opportunities in biotechnological research, so it is important to analyse the possibilities for their cryopreservation in order to establish a biobank. The aim of this experiment was to determine the effect of culture conditions and vitrification on rabbit parthenogenetic embryos. Parthenotes were cultured under in vivo and in vitro conditions until day 3 (late morula/early blastocyst), when they were vitrified. Immediately after warming, they were newly cultured under in vivo and in vitro conditions till day 6 (blastocyst stage). Both culture conditions showed similar late morula/early blastocyst (0.39±0.056 vs. 0.46±0.043, for in vivo and in vitro, respectively) and blastocyst rates (0.12±0.068 vs. 0.13±0.070, for in vivo and in vitro, respectively). However, no parthenote was recovered when a combination of culture conditions was performed. To our best knowledge, this is the first demonstration of the ability of rabbit parthenogenetic embryos to develop after vitrification, with similar embryo development after in vivo or in vitro culture. Nevertheless, our results highlight the importance of culture conditions on the morphology of parthenote embryos. Therefore, we have described that special attention should be paid on culture conditions to generate parthenote embryos, with a view to their subsequent use, for example in embryonic stem cell production.Statement of funding: This work was supported by funds from the Generalitat Valenciana Research Programme (Prometeoll 2014/036).Naturil Alfonso, C.; Jiménez Trigos, ME.; Vicente Antón, JS.; Marco Jiménez, F. (2015). Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification. Cryobiology. 71(1):91-96. doi:10.1016/j.cryobiol.2015.05.003S919671

Nuevas metodologías docentes para un nuevo mundo laboral

[EN] The competences and skills to acquire by the student have evolved, so the educational methodology must also evolve. Society demands professionals with competences and skills in information and communication technologies (ICT), further a critical and deductive thinking. The goal of this current paper is the description of a teaching project that proposes the implementation of flipped classroom in combination with ICT and learning stimulating methodologies, such as gamification. The teaching project will be applied in the subject: Animal and Human Physiology (Degree in Biotechnology, Universitat Piltècnica de València), which includes both “Alto Rendimiento Académico” (English group) and Spanish group. Five activities have been designed to achieve the transversal competences selected for this subject. The teacher, based on the indicators of achievement and competence evidences, will be able to evaluate the effectiveness of the project, comparing the degree of achievement of the skills prior or postproject implementation. Furthermore, the final mark will also be compared with previous years, in order to test whether the innovative educational strategy has had a relevant impact on the final grade of the subject.[ES] Las competencias y habilidades a adquirir por parte del estudiante han evolucionado, por lo que la metodología educativa también debe evolucionar. La sociedad demanda profesionales con competencias y habilidades en tecnologías de la información y la comunicación (TIC) y que demuestren un pensamiento crítico y deductivo. El objetivo del presente trabajo es la descripción de un proyecto docente que propone la implantación de la clase inversa en combinación con TIC y metodologías estimuladoras del aprendizaje, como la gamificación. El proyecto docente se aplicará en la asignatura de Fisiología Animal y Humana (Grado en Biotecnología; Universitat Piltècnica de València), tanto en el grupo Alto Rendimiento Académico (lengua vehicular inglés) como en un segundo grupo donde la lengua vehicular es el castellano.Han sido propuestas 5 actividades diseñadas para alcanzar las competencias transversales propuestas en la asignatura. El docente, basado en los indicadores de consecución y las evidencias de competencia, evaluará la efectividad del proyecto, comparando el grado de adquisición de las competencias previo o post-implantación del proyecto. Además, también se comparará la calificación final respecto a años anteriores, testando así si la innovadora estrategia educativa ha tenido un impacto en la nota final de la asignatura.Este proyecto ha sido financiado por el Vicerrectorado de Estudios, Calidad y Acreditación de la Universidad Politécnica de Valencia (UPV) en el marco del Programa Proyectos de Innovación y Mejora Educativa (PIME/2017/B/010) y la Escuela de Ingeniería Agrícola y Medio Ambiente ( ETSIAMN) de la Universitat Politècnica de València (UPV).Sánchez Peñaranda, D.; Naturil Alfonso, C.; Marco Jiménez, F.; Vicente Antón, JS. (2018). Nuevas metodologías docentes para un nuevo mundo laboral. En IN-RED 2018. IV Congreso Nacional de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 1193-1203. https://doi.org/10.4995/INRED2018.2018.88681193120

Mala gestión del tiempo en los estudiantes universitarios: efectos de la procrastinación

[EN] Academic procrastination is a fact related to the delay or postpone of academic work until last minute. This phenomenon is evident in a vast majority of university students, and its occurrence is increasing. In order to analyse possible causes and/or solutions, we studied if longer time for accomplishing an assignment incentives or avoids procrastination among university students. Results showed that both short and long time-frame groups tended to procrastinate in the same way. Additionally, academic grades did not revealed differences between groups, as the procrastination was the same between groups. Thus, this study shows that even with longer period of time to accomplish a task, university students tend to procrastinate, and thus seem to have a negative effect on their assignment grades. Therefore, it seems a current problem and measures should be developed in order to solve it[ES] La procrastinación académica es un término que hace referencia a la postergación o posposición de cualquier actividad o tarea académica para el último minuto. Este fenómeno prevalece de forma mayoritaria entre los estudiantes universitarios, y su incidencia parece ir en aumento. Con el fin de analizar las posibles causas y/soluciones a la procrastinación, en este trabajo se estudió si un mayor tiempo para la elaboración y/o desarrollo de las actividades y tareas universitarias impedía o incentivaba la procrastinación entre los estudiantes universitarios. Los resultados mostraron que tanto los grupos con menor tiempo para el desarrollo de tareas, como los grupos con un mayor tiempo tendían a procrastinar de la misma manera. Además, las notas medias de las tareas no mostraron diferencias entre los grupos dado que la procrastinación fue la misma en ambos grupos. Por ello, este estudio muestra que incluso con periodos más prolongados para la elaboración de la actividad, los estudiantes universitarios tienden a procrastinar, y este hecho parece tener un efecto negativo sobre la nota de la actividad. En conclusión, la procrastinación es un grave problema en el sistema universitario actual y deberían tomarse medidas para solucionarlo.Este proyecto ha sido financiado por el Vicerrectorado de Estudios, Calidad y Acreditación de la Universidad Politécnica de Valencia (UPV) en el marco del Programa Proyectos de Innovación y Mejora Educativa (PIME/2017/B/010) y la Escuela de Ingeniería Agrícola y Medio Ambiente ( ETSIAMN) de la Universitat Politècnica de València (UPV).Naturil Alfonso, C.; Peñaranda, D.; Marco Jiménez, F.; Vicente Antón, JS.; Peñaranda (2018). Mala gestión del tiempo en los estudiantes universitarios: efectos de la procrastinación. En IN-RED 2018. IV Congreso Nacional de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 1268-1275. https://doi.org/10.4995/INRED2018.2018.8874OCS1268127

Study of failures in a rabbit line selected for growth rate

[EN] Selection for growth rate is negatively related with reproductive fitness. The aim of this work was to analyse the causes of fertility failure in rabbit does selected for growth rate and characterised for reproductive deficiencies (line R). In the experiment, 82 does were divided into 2 groups: naturally mated (NM) and artificially inseminated (AI), to relate luteinizing hormone (LH) concentration with ovulation induction and pregnancy rate by laparoscopic determination. Additionally, in 38 of these females ovulation rate and metabolites determination (leptin, NEFA, BOHB and glucose) were analysed and perirenal fat thickness measurement and live body weight (LBW) determined. The results showed that all ovulated does (both NM and AI) presented higher concentrations of LH than non-ovulated females. In addition, non-ovulated females showed high levels of leptin and BOHB, as well as LBW. Females from line R have an inherit reduced fertility due to ovulation failure as a consequence of a reduction in LH release, which could be explained by a heavier body weight and higher leptin concentrations.This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT). Carmen Naturil-Alfonso was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2013/296). English text version was revised by N. Macowan English Language Service.Naturil Alfonso, C.; Lavara García, R.; Millán, P.; Rebollar, P.; Vicente Antón, JS.; Marco Jiménez, F. (2016). Study of failures in a rabbit line selected for growth rate. World Rabbit Science. 24(1):47-53. https://doi.org/10.4995/wrs.2016.4016SWORD475324

Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al.This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2012). Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS ONE. 7(12):1-11. https://doi.org/10.1371/journal.pone.0051271S111712Harness, J. 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Proceedings of the National Academy of Sciences, 100(Supplement 1), 11911-11916. doi:10.1073/pnas.2034195100Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., … Liu, L. (2009). Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells. Stem Cells, 27(9), 2136-2145. doi:10.1002/stem.158Sritanaudomchai, H., Ma, H., Clepper, L., Gokhale, S., Bogan, R., Hennebold, J., … Mitalipov, S. (2010). Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells. Human Reproduction, 25(8), 1927-1941. doi:10.1093/humrep/deq144Fang, Z. F., Gai, H., Huang, Y. Z., Li, S. G., Chen, X. J., Shi, J. J., … Sheng, H. Z. (2006). Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Experimental Cell Research, 312(18), 3669-3682. doi:10.1016/j.yexcr.2006.08.013Wang, S., Tang, X., Niu, Y., Chen, H., Li, B., Li, T., … Ji, W. (2007). Generation and Characterization of Rabbit Embryonic Stem Cells. Stem Cells, 25(2), 481-489. doi:10.1634/stemcells.2006-0226Piedrahita, J. A., Anderson, G. B., & BonDurant, R. H. (1990). On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos. Theriogenology, 34(5), 879-901. doi:10.1016/0093-691x(90)90559-cNaturil-Alfonso, C., Saenz-de-Juano, M. D., Peñaranda, D. S., Vicente, J. S., & Marco-Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science, 127(3-4), 222-228. doi:10.1016/j.anireprosci.2011.08.005Besenfelder, U., Strouhal, C., & Brem, G. (1998). A Method for Endoscopic Embryo Collection and Transfer in the Rabbit. Journal of Veterinary Medicine Series A, 45(1-10), 577-579. doi:10.1111/j.1439-0442.1998.tb00861.xMehaisen, G. M. K., Viudes-de-Castro, M. P., Vicente, J. S., & Lavara, R. (2006). In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does. Theriogenology, 65(7), 1279-1291. doi:10.1016/j.theriogenology.2005.08.007Bilodeau-Goeseels, S., & Schultz, G. A. (1997). Changes in Ribosomal Ribonucleic Acid Content Within in Vitro-produced Bovine Embryos1. Biology of Reproduction, 56(5), 1323-1329. doi:10.1095/biolreprod56.5.1323Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M., & Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18), 3674-3676. doi:10.1093/bioinformatics/bti610Edgar, R. (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Research, 30(1), 207-210. doi:10.1093/nar/30.1.207Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.019Llobat, L., Marco-Jiménez, F., Peñaranda, D., Saenz-de-Juano, M., & Vicente, J. (2011). Effect of Embryonic Genotype on Reference Gene Selection for RT-qPCR Normalization. Reproduction in Domestic Animals, 47(4), 629-634. doi:10.1111/j.1439-0531.2011.01934.xLiu, N., Enkemann, S. A., Liang, P., Hersmus, R., Zanazzi, C., Huang, J., … Liu, L. (2010). Genome-wide Gene Expression Profiling Reveals Aberrant MAPK and Wnt Signaling Pathways Associated with Early Parthenogenesis. Journal of Molecular Cell Biology, 2(6), 333-344. doi:10.1093/jmcb/mjq029Abdoon, A. S., Ghanem, N., Kandil, O. M., Gad, A., Schellander, K., & Tesfaye, D. (2012). cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos. Theriogenology, 77(6), 1240-1251. doi:10.1016/j.theriogenology.2011.11.004Labrecque, R., & Sirard, M.-A. (2011). 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Cell Research, 21(3), 466-473. doi:10.1038/cr.2011.15Mamo, S., Gal, A., Polgar, Z., & Dinnyes, A. (2008). Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos. BMC Molecular Biology, 9(1), 67. doi:10.1186/1471-2199-9-67Navarrete Santos, A., Tonack, S., Kirstein, M., Pantaleon, M., Kaye, P., & Fischer, B. (2004). Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts. Reproduction, 128(5), 517-526. doi:10.1530/rep.1.0020

Parthenogenic blastocysts cultured under in vivo conditions exhibited proliferation and differentiation expression genes similar to those normal embryos

Parthenotes embryos offer multiple possibilities in biotechnological investigations, such as stem cell research. However, there is still a dearth of knowledge of this kind of embryos. In this study, we analyzed development and ploidy in parthenotes under in vitro and in vivo culture conditions. Subsequently, using real-time PCR, we analyzed the expression of factor OCT-4, Vascular Endothelial Growth Factor, Epidermal Growth Factor Receptor 3 and Transforming Growth Factor )2 genes to compare the kind of embryo at the blastocyst stage. Development and implantation of parthenotes embryos were describe after transfer at day 10 of pregnancy. Parthenotes showed similar blastocyst development for both culture conditions and that most of the parthenotes produced were diploid. However, parthenotes developed under in vivo conditions showed similar mRNA expression of OCT-4, VEGF and TGF- 2 to 5 and 6 day old blastocysts. While, parthenotes developed under in vitro conditions had altered the expression pattern of these genes, except for erbB3 mRNA. Finally, transferred parthenotes had the ability to implant but showed severe growth retardation and lesser size. This is the first demonstration of the influence of culture conditions on parthenotes mRNA expression. Our study highlights the importance of culture conditions in the subsquent uses of parthenotes such as the production of stem cell lines.Naturil Alfonso, C. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibited proliferation and differentiation expression genes similar to those normal embryos. http://hdl.handle.net/10251/15572Archivo delegad

Influence of zona pellucida thickness on fertilization, embryo implantation and birth

Defective sperm-zona pellucida binding and penetration are the main causes of IVF failure. The purpose of this study was to evaluate the effect of zona pellucida thickness in fertilization failure and test the influence of zona pellucida thickness on implantation and birth in rabbits. Embryos and oocytes were collected from 72 females on Day 2 post-insemination. A total of 559 normal embryos were recovered; 402 embryos were transferred by laparoscopy and 157 embryos were used to measure the zona pellucida thickness using the ImageJ program. Laparoscopies were also performed on all does at Day 12 of gestation to record the number of implanted embryos. Litter size at birth was recorded. The mean zona pellucida thickness of the 157 embryos and of the 64 control group oocytes (18.3 ± 0.2 and 18.5 ± 0.3 ¿m, respectively) was significantly less than the zona pellucida thickness of the 74 failed fertilization oocytes (19.2 ± 0.3 ¿m). The probabilities of the regression coefficient being positive were 0.72 and 0.74 for implantation and birth, respectively, and the subsequent means of the coefficient were 2.92 and 0.03 for implantation and birth, respectively. In conclusion, the zona pellucida thickness has an important influence on in vivo fertilization and implantation processes, but not on birth. © 2012 Elsevier B.V.This research was supported by the Valencian Regional Government research programme (Prometeo 2009/125) and the Spanish Research Projects (CICYTAGL2008-03274). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.Marco Jiménez, F.; Naturil Alfonso, C.; Jiménez Trigos, ME.; Lavara García, R.; Vicente Antón, JS. (2012). Influence of zona pellucida thickness on fertilization, embryo implantation and birth. Animal Reproduction Science. 1-2(132):96-100. https://doi.org/10.1016/j.anireprosci.2012.04.008S961001-213