74 results on '"Aguila L"'
Search Results
2. Effect of sperm treatment with lysolecithin on in vitro outcomes of equine intracytoplasmic sperm injection
- Author
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Aguila, L., primary, Cabrera, P., additional, Arias, ME., additional, Silva, M., additional, and Felmer, R., additional
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- 2024
- Full Text
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3. 141 Enhanced developmental potential of bovine embryos generated by piezo-intracytoplasmic sperm injection with sorted capacitated spermatozoa by fluorescence-activated cell sorting
- Author
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Castro, M., primary, Aguila, L., additional, Arias, M. E., additional, and Felmer, R., additional
- Published
- 2023
- Full Text
- View/download PDF
4. Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?
- Author
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Sepúlveda, B., Arias, M. E., Aguila, L., Zambrano, F., Sánchez, R., and Felmer, R.
- Published
- 2018
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- View/download PDF
5. P359 Optimized methods to model Mycoplasma genitalium reproductive tract infection in pig-tailed macaques
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Aguila, L, primary, Patton, D, additional, and Wood, G, additional
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- 2021
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6. O01.5 Antigenic variation in a primate model of persistent Mycoplasma genitalium infection
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Wood, G, primary, Aguila, L, additional, and Patton, D, additional
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- 2021
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- View/download PDF
7. Efeito do sistema de cultivo e arranjo de plantas de soja sobre a produtividade de cultivar adaptada ao ambiente de Terras Baixas de clima temperado
- Author
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AGUILA, L. S. H. D., COSTA, V. S. da, VASQUES, S. R., SILVA, S. M. da, VIEIRA, P. A., SINNEMANN, C., CAMPOS, A. D. S. de, ÁVILA, A. F., LEITE, T. M., SOARES, V. de A., ALVES, A. C. de O., ALVES, B. R. S., LILIA SICHMANN HEIFFIG DEL AGUILA, CPACT, VAGNER SCOUTO DA COSTA, UFPEL, SHAMILY RODRIGUES VASQUES, IFSul/CAVG, SABRINA MONCKS DA SILVA, UFPEL, PAOLA ACOSTA VIEIRA, UFPEL, CAMILA SINNEMANN, UFPEL, ALEXSSANDRA DAYANE SOARES DE CAMPOS, UFPEL, AMANDA FISS ÁVILA, IFSul/CAVG, THALIA MARTINS LEITE, IFSul/CAVG, VANESSA DE AVILA SOARES, IFSul/CAVG, ANA CAROLINA DE OLIVEIRA ALVES, UFPEL, and BRUNA REGINA SOUZA ALVES, UFPEL.
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Solo ,Glycine Max ,Bioma Pampa ,Soja ,Terras baixas - Abstract
Os planossolos das Terras Baixas do Rio Grande do Sul caracterizam-se por apresentar horizonte A superficial e horizonte B com capacidade de percolação muito baixa, o que provoca longos períodos de encharcamento após chuvas e prejudica o desenvolvimento e a produtividade da soja. Para evitar esses problemas, são necessários sistemas de drenagem adequados e o aprimoramento de sistemas de preparo do solo e de cultivo que proporcionem faixas mais secas na lavoura. O presente trabalho tem por objetivo determinar o arranjo espacial de plantas e o sistema de cultivo de soja mais adequado para as Terras Baixas do Rio Grande do Sul. Dois experimentos foram conduzidos na Estação Experimental Terras Baixas da Embrapa Clima Temperado, em Capão do Leão/RS. O primeiro deles (safra 2016/17) diferenciou o potencial produtivo de cultivares indicadas para terras baixas, sendo a cultivar BMX Icone RR a mais produtiva, com 4073 kg ha-1 de grãos enquanto outras 10 cultivares produziram em média 3021 kg ha-1. No segundo experimento (safra 2018/19), a cultivar BMX Icone foi adotada. Foram avaliados 2 manejos de solo (preparo convencional e preparo com sulco/camalhão) e 3 arranjos de plantas (230, 330 e 430 mil plantas ha-1). Foram avaliadas a altura de plantas; altura de inserção da primeira vagem; nº de ramificações da haste principal; peso de grãos; número de grãos e vagens por planta; e produtividade de grãos. O sulco-camalhão com 330 mil plantas ha-1 com espaçamento entrelinhas 35 / 60 cm, propiciou melhor desempenho geral da cultura e melhor produtividade. Made available in DSpace on 2020-11-20T09:13:42Z (GMT). No. of bitstreams: 1 16953-43703-1-PB.pdf: 527668 bytes, checksum: 183147c8d68604554a6f9d452e70d36c (MD5) Previous issue date: 2020 Título em inglês: Effect of the cultivation system and spatial arrangement of soybean plants on the productivity of cultivar adapted to the environment of lowland areas.
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- 2020
8. Produtividade da soja no Rio Grande do Sul: genética ou manejo?
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CONCENCO, G., AGUILA, L. S. H. D., VERNETTI JUNIOR, F. de J., GERMANI CONCENCO, CPACT, LILIA SICHMANN HEIFFIG DEL AGUILA, CPACT, and FRANCISCO DE JESUS VERNETTI JUNIOR, CPACT.
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Soja - Abstract
Made available in DSpace on 2017-10-20T09:28:39Z (GMT). No. of bitstreams: 1 GermaniConcencoArtigoSojaEmbrapaCULTIVAR.pdf: 194075 bytes, checksum: 5c7fc8f173d23a541adbd13bc2857bf8 (MD5) Previous issue date: 2017-10-19
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- 2017
9. Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?
- Author
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Sepúlveda, B., primary, Arias, M. E., additional, Aguila, L., additional, Zambrano, F., additional, Sánchez, R., additional, and Felmer, R., additional
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- 2017
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10. Effect of sperm pretreatment with glutathione and membrane destabilizing agents lysolecithin and Triton X-100, on the efficiency of bovine intracytoplasmic sperm injection
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Zambrano, F, primary, Aguila, L, additional, Arias, ME, additional, Sanchez, R, additional, and Felmer, R, additional
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- 2017
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11. Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?
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Aguila, L., Sepúlveda, B., Arias, M. E., Felmer, R., Zambrano, F., and Sánchez, R.
- Subjects
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CATTLE reproduction , *SPERMATOZOA , *REACTIVE oxygen species , *BOS , *EMBRYOLOGY - Abstract
Summary: In assisted reproductive techniques, it is essential to perform a sperm selection to obtain spermatozoa with high motility and membrane integrity for in vitro fertilisation (IVF) and high‐DNA integrity for intracytoplasmic sperm injection (ICSI). In this study, we evaluated whether Isolate
® was a suitable substitute for Percoll® for assisted reproductive techniques. Commercial cryopreserved bovine semen was used after selection in both gradients, and plasma and acrosome membrane integrity, reactive oxygen species (ROS) levels, DNA integrity and mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. Motility parameters were also evaluated by CASA system. A similar percentage of spermatozoa with intact plasma membrane, acrosome integrity and high ΔΨm was observed in both sperm selection methods, but only Percoll® showed higher percentage of spermatozoa with intact plasma and acrosome membrane compared to the post‐thawing group. No differences were observed in the motility, ROS, DNA fragmentation and on the in vitro embryo production in all experimental groups. In conclusion, the selection of bovine spermatozoa with Isolate® generates spermatozoa with similar quality parameters and embryonic development compared to Percoll® providing a suitable alternative sperm selection method for assisted reproductive techniques in this species. [ABSTRACT FROM AUTHOR]- Published
- 2018
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12. Indutor de resistência na pós-colheita de laranja 'Salustiana'
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TOMAZETTI, T. C., ROSSAROLLA, M. D., COPATTI, A. S., MONTEIRO, A. L., RICHI, P. S., AGUILA, L. S. H. D., AGUILA, J. S. D., Tomazetti, T.C., UNIPAMPA, ROSSAROLLA, M. D., UNIPAMPA, Copatti, A. S., UNIPAMPA, Monteiro, A. L., UFPEL, Righi, P. S, UFPEL, LILIA SICHMANN HEIFFIG DEL AGUILA, CPACT, and Juan Saavedra del Aguila, UNIPAMPA.
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Osbeck ,Citrus sinensis L ,Metabolismo sucundário ,Mofo Verde - Abstract
Made available in DSpace on 2014-03-11T23:27:16Z (GMT). No. of bitstreams: 1 RevIberTecnologiaPostcosecha14220132.pdf: 258553 bytes, checksum: 7d7f21d123787964350b8d7477df09a9 (MD5) Previous issue date: 2014-03-10
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- 2013
13. Bicarbonato de sódio (NaHCO3) mantém a firmeza de frutos de bergamota 'Clementina' inoculados com Penicillium digitatum
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CAMPONOGARA, T. T., ROSSAROLLA, M. D., AGUILA, L. S. H. D., AGUILA, J. S. D., RADMANN, E. B., TOMAZETTI, Tiago Camponogara, UNIPAMPA, ROSSAROLLA, Márcia Denise, UNIPAMPA, LILIA SICHMANN HEIFFIG DEL AGUILA, CPACT, SAAVEDRA DEL AGUILA, Juan, UNIPAMPA, and RADMANN, Elizete Beatriz, UNIPAMPA.
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Citrus sinensis (L.) Osbeck ,Secondary metabolism - Abstract
Resumos do VIII Congresso Brasileiro de Agroecologia - Porto Alegre/RS - 25 a 28/11/2013.
- Published
- 2013
14. Determination of optimal ethylene concentration applied in postharvest mango fruit
- Author
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AGUILA, J. S. D., LIMA, F. V., GAZZOLA, A. E., ORTEGA, E. M. M., AGUILA, L. S. H. D., KLUGE, R. A., DEL AGUILA, Juan Saavedra, INIPAMPA, Lima, F.V, GAZZOLA, A. E., ORTEGA, Edwin Moisés Marcos, LILIA SICHMANN HEIFFIG DEL AGUILA, CPACT, and KLUGE, Ricardo Alfredo.
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Magifera indica L ,Primary metabolism ,Tropical fruit ,Climacteric fruit - Abstract
Made available in DSpace on 2014-03-11T23:26:44Z (GMT). No. of bitstreams: 1 RevIberTecnologiaPostcosecha14220131.pdf: 321110 bytes, checksum: cf82e4c174ebe844afbb103e7b5eb369 (MD5) Previous issue date: 2014-03-10
- Published
- 2013
15. O ácido salicílico em pré-colheita influencia o controle pós-colheita de Penicillium digitatum de laranja 'Salustiana'?
- Author
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ROSSAROLLA, M. D., TOMAZETTI, T. C., COPATTI, A. S., MONTEIRO, A. de M., RIGHI, P. S., AGUILA, L. S. H. D., AGUILA, J. S. D., MÁRCIA DENISE ROSSAROLLA, TIAGO CAMPONOGARA TOMAZETTI, ANDRIO SPILLER COPATTI, ALINE DE MELO MONTEIRO, LILIA SICHMANN HEIFFIG DEL AGUILA, CPACT, JUAN SAAVEDRA DEL AGUILA., Márcia Denise Rossarolla, UNIPAMPA, Tiago Camponogara Tomazetti, UNIPAMPA, Andrio Spiller Copatti, UNIPAMPA, Aline de Melo Monteiro, UNIPAMPA, Pércio Sanchez Righi, UFPel, and Juan Saavedra del Aguila, UNIPAMPA.
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Osbeck ,Fenol ,Citrus Sinensis ,Citrus sinensis (L.) Osbeck ,Metabolismo secundário - Published
- 2012
16. 216 EFFECT OF SPERM PRETREATMENT WITH LYSOLECITHIN AND Triton X-100 ON PRONUCLEAR FORMATION AND QUALITY OF BOVINE EMBRYOS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION
- Author
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Zambrano, F., primary, Arias, M. E., additional, Vargas, T., additional, Aguila, L., additional, and Felmer, R., additional
- Published
- 2016
- Full Text
- View/download PDF
17. AB0816 Altered Sleep Patterns, Fatigue, Anxiety and Depression Levels are Important Features Among Patients with Psoriatic Arthritis
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Arancibia Aguila, L., primary, Medeiros, A.C., additional, Gonçalves, C.R., additional, Sampaio Barros, P.D., additional, and Goldenstein Schainberg, C., additional
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- 2015
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18. 318 EFFECT OF SPERM PRETREATMENT WITH ISOBUTYLMETHYLXANTHINE AND METHYL-β-CYCLODEXTRIN ON THE EFFICIENCY OF BOVINE INTRACYTOPLASMIC SPERM INJECTION
- Author
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Aguila, L. M., primary, Arias, M. E., additional, Sanchez, R. S., additional, Vargas, T. C., additional, Zambrano, F. A., additional, and Felmer, R. F., additional
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- 2015
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19. Optical arrangement coupled to a radial shear cyclic interferometer to generate simultaneously two interferograms with a phase shift of π/ 2
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Casco Vásquez, J. F., primary, Robledo Sánchez, C. I., additional, Ortiz Gutiérrez, M., additional, Arévalo Aguila, L. M., additional, and Nanco Hernandez, P., additional
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- 2011
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20. Effects of age on free fatty acid induced insulin resistance
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Kirwan, J., Krishnan, R., Marchetti, C., Williamson, D., del Aguila, L., O'Gorman, D., and Stetzer, B.
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Geriatrics -- Research ,Insulin resistance -- Physiological aspects ,Insulin resistance -- Causes of ,Fatty acids -- Physiological aspects ,Fatty acids -- Influence ,Muscles -- Physiological aspects ,Health ,Seniors - Abstract
Age-related insulin resistance may be due to the suppressive effects of elevated free fatty acids (FFA) on glucose oxidation in skeletal muscle (i.e., the Randle Cycle). To examine this question, two-stage euglycemic (5.0 mM) hyperinsulinemic (40 mU.m2.min-1) clamps were performed on nine older obese subjects with abnormal glucose tolerance (67 + 2 yr; 83.4 + 2.8 kg; BMI, 29.2 + 0.3 kg/m2) and nine healthy young subjects (24 + 2 yr; 65.9 + 4.1 kg; BMI, 21.3 +1.1 kg/m2) with normal glucose tolerance. Glucose and insulin were infused during the first stage of the clamp (120 min), to measure insulin mediated glucose infusion rate (GIR). Intralipid and heparin infusions were added during the second stage (300 min), to measure FFA suppression of insulin mediated GIR and glucose oxidation. Basal FFA (0.75 + 0.05 vs 0.51 + 0.09 mEq/L, p
- Published
- 2002
21. Symmetry reductions for a nonlinear diffusion-absorption equation in two spatial dimensions
- Author
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Gandarias, M. L, primary and Aguila, L. del, additional
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- 1998
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22. FREE FATTY ACID SUPPRESSION OF INSULIN MEDIATED GLUCOSE METABOLISM IN OLDER OVERWEIGHT INDIVIDUALS
- Author
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Lewis, R., primary, Williamson, D. L., additional, O'Gorman, D. J., additional, del Aguila, L., additional, Krishnan, R. K., additional, and Kirwan, J. P., additional
- Published
- 1998
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23. Optical arrangement coupled to a radial shear cyclic interferometer to generate simultaneously two interferograms with a phase shift of / 2
- Author
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Casco Vásquez, J. F., Robledo Sánchez, C. I., Ortiz Gutiérrez, M., Arévalo Aguila, L. M., and Nanco Hernandez, P.
- Abstract
A radial displacement cyclic interferometer parallel beams is used to generate phase shifts. Simultaneously engages an architecture that generates the same moment two interferograms with a phase shift of pi / 2 between them. This phase shift is obtained using optical components. Unlike the arrangements that use a grid in the Fourier plane, where the distance between interference patterns obtained are not adjustable, but is determined by the length of the grid, in the configuration presented, is sufficient to shift one of the reflecting mirrors in order to control the separation between interferograms.. A phase object is used. Interferometric arrangement is presented, and the results obtained experimentally.
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- 2011
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24. 35th Annual Meeting of the European Association for the Study of Diabetes
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Melander, A., Olsson, J., Lindberg, G., Salzman, A., Howard, T., Stang, P., Lydick, E., Emslie-Smith, A., Boyle, D. I. R., Evans, J. M. M., Macdonald, T. M., Bain, J., Sullivan, F., Juhl, C., Pørksen, N., Sturis, J., Hollingdal, M., Pincus, S., Veldhuis, J., Dejgaard, A., Schmitz, O., Kristensen, J. S., Frandsen, K. B., Bayer, Th., Müller, P., Dunning, B. E., Paladini, S., Gutierrez, C., Deacon, R., Valentin, M., Grunberger, G., Weston, W. M., Patwardhan, R., Rappaport, E. B., Sargeant, L. A., Wareham, N. J., Khaw, K. T., Zethelius, Björn, Lithell, Hans, Hales, C. Nicholas, Berne, Christian, Lakka, H.-M., Oksanen, L., Tuomainen, T.-P., Kontula, K., Salonen, J. T., Dekker, J. M., de Boks, P., de Vegt, F., Stehouwer, C. D. A., Nijpels, G., Bouter, L. M., Heine, R. J., Bruno, G., Cavallo-Perin, P., Bargero, G., D’Errico, N., Borra, M., Macchia, G., Pagano, G., Newton, R. W., Ruta, D. A., New, J. P., Wallace, C., Roxburgh, M. A., Young, R. J., Vaughan, N. J. A., Elliott, P., Brennan, G., Devers, M., MacAlpine, R., Steinke, D., Lawson, D. H., Decallonne, B., Casteels, K., Gysemans, C., Bouillon, R., Mathieu, C., Linn, Thomas, Strate, Christine, Schneider, Kerstin, Funda, D. P., Jirsa, M., Kozáková, H., Kaas, A., Kofronová, O., Tlaskalová-Hogenová, H., Buschard, K., Wanka, H., Hartmann, A., Kuttler, B., Rasmussen, S. B., Sørensen, T. S., Markholst, H., Petersen, J. S., Karounos, D., Dyrberg, T., Mabley, J. G., Haskó, G., Szabó, C., Seissler, J., Nguyen, T. B. T., Steinbrenner, H., Scherbaum, W. A., Cipriani, R., Gabriele, A., Sensi, M., Guidobaldi, L., Pantellini, F., Cerrito, M. G., Scarpa, S., Di Mario, U., Morano, S., Ceolotto, G., Iori, E., Baritono, E., Del Prato, S., Semplicini, A., Trevisan, R., Zerbini, G., Meregalli, G., Asnaghi, V., Tentori, F., Maestroni, A., Mangili, R., Marescotti, C., Vedovato, M., Tiengo, A., Tadjieva, J., Mankovsky, B. N., Van Aken, S., Raes, A., Vande Walle, J., Matthys, D., Craen, M., Hansen, H. P., Lund, S. S., Rossing, P., Jensen, T., Parving, H.-H., Andersen, S., Tarnow, L., Hansen, B. V., Trautner, C., Haastert, B., Ennenbach, N., Willich, S., Tabák, Á. Gy., Orchard, T. J., Spranger, J., Preissner, K. T., Schatz, H., Pfeiffer, A., Cantón, A., Burgos, R., Hernández, C., Lecube, A., Mesa, J., Segura, R. M., Mateo, C., Simó, R., Fathallah, L., Greene, D. A., Obrosova, I., Gilbert, R. E., Kelly, D. J., Cox, A. J., Berka-Wilkinson, J. L., Taylor, H. R., Panagiotopoulos, S., Lee, V., Jerums, G., Cooper, M. E., Hitman, G. A., Aganna, E., Ogunkolade, W. B., Rema, M., Deepa, R., Shanthi-Rani, C. S., Barakat, K., Kumarajeewa, T. R., Cassell, P. G., McDermott, M. F., Mohan, V., Ways, K., Bursell, S., Devries, T., Woodworth, J., Alatorre, C., King, G., Aiello, L. P., Karisen, A. E., Pavlovic, D., Nielsen, K., Jensen, J., Andersen, H. U., Pociot, F., Mandrup-Poulsen, T., Eizirik, D. L., Nerup, J., Lortz, S., Tiedge, M., Lenzen, S., Lally, F. J., Bone, A. J., Darville, M. I., Ho, Y.-S., Sternesjö, J., Sandler, S., Chen, M.-C., Schuit, F., Pipeleers, D. G., Merezak, S., Hardikar, A., Hoet, J. J., Remacle, C., Reusens, B., Bréant, B., Garofano, A., Czernichow, P., Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Nakano, R., Komeda, K., Eto, K., Tobe, K., Kimura, S., Kadowaki, T., Ide, T., Murakami, K., Tsunoda, M., Mochizuki, T., Ozanne, S. E., Nave, B. T., Wang, C. L., Dorling, M. W., Petry, C. J., Koopmans, S. J., van der Bent, C., Que, I., Radder, J. K., Sebokova, E., Sana, A. K., Klimes, I., Ruderman, N., Morviducci, L., Pastore, L., Morelli, S., Sagratella, E., Zorretta, D., Buongiomo, A., Tamburrano, G., Giaccari, A., Martinenghi, Sabina, De Angelis, Gabriella Cusella, Ravasi, Flavio, Bifari, Francesco, Bordignon, Claudio, Falqui, Luca, Kessler, A., Dransfeld, O., Sasson, S., Tomas, E., Zorzano, A., Eckel, J., Thorsby, P., Rosenfalck, A. M., Kjems, L., Hanssen, K. F., Madsbad, S., Birkeland, K. I., Hamilton-Wessler, M., Markussen, J., Bergman, R. N., Melki, V., Hanaire-Broutin, H., Bessières-Lacombe, S., Tauber, J.-P., Home, P. D., Lindholm, A., Riis, A., Rosenstock, J., Schwartz, S., Clark, C., Edwards, M., Donley, D., Swift, P., Mortensen, H. B., Lynggaard, H., Hougaard, P., Cull, C. A., Neil, H. A. W., Frighi, V., Manley, S. E., Holman, R. R., Turner, R. C., Steiner, G., Davis, W. A., Weeraratna, T., Bruce, D. G., Davis, T. M. E., Vergès, B., Duvillard, L., Pont, F., Florentin, E., Gambert, Ph., Benko, B., Ljubić, S., Turk, Z., Granić, M., März, W., Wollschläger, H., Klein, G., Neiss, A., Wehling, M., Huxtable, S. J., Saker, P. J., Walker, M., Frayling, T. M., Levy, J. C., O’Rahilly, S., Hattersley, A. T., McCarthy, M. I., Orecchio, A., Giacchini, A., Dominici, R., Canettieri, G., Trinti, B., Zani, M., Andreoli, M., Sciacchitano, S., de Silva, A. M., Whitecross, K., Pasco, J., Kotowicz, M., Nicholson, G., Zimmet, P., Boyko, E. J., Collier, G. R., Frittitta, L., Pizzuti, A., Argiolas, A., Graci, S., Goldfine, I. D., Bozzali, M., Ercolino, T., Costanzo, B., Iacoviello, L., Tassi, V., Trischitta, V., Wauters, M., Rankinen, T., Mertens, I., Chagnon, M., Bouchard, C., Van Gaal, L., Sivenius, K., Valve, R., Hakkarainen, V., Niskanen, L., Laakso, M., Uusitupa, M., Beridze, N., Japaridze, M., Kurashvili, R., Dundua, M., Kebuladze, G., Kazakhashvili, N., Offley-Shore, B., Thomas, B., Ghebremeskel, K., Crawford, M., Lowy, C., Eriksson, Ulf J., Martin Simán, C., Wisse, Bert, Gittenberger-de Groot, Adriana C., Wentzel, P., Eriksson, U. J., Wender-Ożegowska, E., Drews, K., Biczysko, R., Bronisz, A., Rość, D., Graczykowska-Koczorowska, A., Kotschy, M., Sokup, A., Kohnert, K. D., Besch, W., Strese, J., Frick, U., Zander, E., Kemer, W., Škrha, J., Kvasnička, J., Kalvodová, B., Hilgertová, J., Schatteman, K., Goossens, F., Scharpé, S., De Leeuw, I., Hendriks, D., Legakis, I. N., Panayiotou, D., Mountokalakis, Th. D., Enderle, M. D., Beckmann, P., Balletshofer, B., Rittig, K., Maerker, E., Volk, A., Meisner, C., Jacob, S., Matthaei, S., Häring, H. U., Rett, K., Ueda, K., Nakagawa, T., Shimajiri, Y., Kokawa, M., Matsumoto, E., Sasaki, H., Sanke, T., Nanjo, K., McKinnon, Caroline M., Macfarlane, Wendy M., Docherty, Kevin, Furukawa, N., Shirotani, T., Kishikawa, H., Kaneko, K., Araki, E., Shichiri, M., Prentki, M., Roduit, R., Susini, S., Buteau, J., Ejrnæs, A. M., Andersen, N. Aa., Osterhoff, M., Möhlig, M., Ortmann, J., Bikashaghi, F., Mayer, C., Bikashagi, F., Ackermans, M. T., Pereira Arias, A. M., Bisschop, P. H. L. T., Endert, E., Sauerwein, H. P., Romijn, J. A., Gastaldelli, A., Baldi, S., Pettiti, M., Natali, A., Frascerra, S., Camastra, S., Toschi, E., Ferrannini, E., Stingl, H., Krssak, M., Bischof, M. G., Krebs, M., Fürnsinn, C., Nowotny, P., Waldhäusl, W., Roden, M., Neeft, M., Meijer, A. 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- 1999
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25. A non-toxigenic strain of aspergillus flavus as a biological control against aflatoxin production in peanuts
- Author
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Calori-Domingues, M. A., Teles, D., Ponce, G. H., Adriano Costa de Camargo, Dias, C. T. S., Da Gloria, E. M., Aguila, L. S. H., and Godoy, I. J.
26. 35th Annual Meeting of the European Association for the Study of Diabetes : Brussels, Belgium, 28 September-2 October 1999
- Author
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Melander, A., Olsson, J., Lindberg, G., Salzman, A., Howard, T., Stang, P., Lydick, E., Emslie-Smith, A., Boyle, D. I. R., Evans, J. M. M., Macdonald, T. M., Bain, J., Sullivan, F., Ad Darts Memo, Morris For The Collaboration, Juhl, C., Porksen, N., Sturis, J., Hollingdal, M., Pincus, S., Veldhuis, J., Dejgaard, A., Schmitz, O., Kristensen, J. S., Frandsen, K. B., Bayer, Th, Muller, P., Dunning, B. E., Paladini, S., Gutierrez, C., Deacon, R., Valentin, M., Grunberger, G., Weston, W. M., Patwardhan, R., Rappaport, E. B., Sargeant, L. A., Wareham, N. J., Khaw, K. T., Zethelius, Bjorn, Lithell, Hans, Hales, C. Nicholas, Berne, Christian, Lakka, H-M, Oksanen, L., Tuomainen, T-P, Kontula, K., Salonen, J. T., Dekker, J. M., Boks, P., Vegt, F., Stehouwer, C. D. A., Nijpels, G., Bouter, L. M., Heine, R. J., Bruno, G., Cavallo-Perin, P., Bargero, G., D Errico, N., Borra, M., Macchia, G., Pagano, G., Newton, R. W., Ruta, D. A., New, J. P., Wallace, C., Roxburgh, M. A., Young, R. J., Vaughan, N. J. A., Elliott, P., Brennan, G., Devers, M., Macalpine, R., Steinke, D., Lawson, D. H., Decallonne, B., Casteels, K., Gysemans, C., Bouillon, R., Mathieu, C., Linn, Thomas, Strate, Christine, Schneider, Kerstin, Funda, D. P., Jirsa, M. Jr, Kozakova, H., Kaas, A., Kofronova, O., Tlaskalova-Hogenova, H., Buschard, K., Wanka, H., Hartmann, A., Kuttler, B., Rasmussen, S. B., Sorensen, T. S., Markholst, H., Petersen, J. S., Karounos, D., Dyrberg, T., Mabley, J. G., Hasko, G., Szabo, C., Seissler, J., Nguyen, T. B. T., Steinbrenner, H., Scherbaum, W. A., Cipriani, R., Gabriele, A., Sensi, M., Guidobaldi, L., Pantellini, F., Cerrito, M. G., Scarpa, S., Di Mario, U., Morano, S., Ceolotto, G., Iori, E., Baritono, E., Del Prato, S., Semplicini, A., Trevisan, R., Zerbini, G., Meregalli, G., Asnaghi, V., Tentori, F., Maestroni, A., Mangili, R., Marescotti, C., Vedovato, M., Tiengo, A., Tadjieva, J., Mankovsky, B. N., Aken, S., Raes, A., Vande Walle, J., Matthys, D., Craen, M., Hansen, H. 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T., Kardos, A., Gingl, Z., Kempler, P., Rudas, L., Lonovics, J., Marchand, M., Stevens, L. K., Tarnas, Gy, Eurodiab Iddm, Study Group, Estrella, F., Christensen, N. J., Keresztes, K., Barna, I., Hermanyi, Zs, Vargha, P., Bonnevie, L., Chanudet, X., Larroque, P., Tutuncu, N. Bascil, Deger, A., Batur, M. K., Yildirir, A., Onalan, O., Aksoyek, S., Kabakciota, G., Erbas, T., Galicka-Latala, D., Surdacki, A., Gerritsen, J., Tenvoorde, B. J., Heethaar, R. M., Tagawa, T. S., Kodama, M., Yoshioka, R., Yamasaki, Y., Didangelos, T., Athyros, V., Kontopoulos, A., Papageorgiou, A., Karamitsos, D., Lacigova, S., Rusavy, Z., Karova, R., Perrild, H., Kay, L., Jorgensen, T., Bien, A. I., Witek, P., Geraldes, Elizabete, Rodrigues, D., Pereira, L., Domenech, A., Leitao, P., Anagnostopoulos, D., Foster, A. V. M., Nag, S., Barsoum, M., Lewis, G., Dunlop, N., Connolly, V., Bilous, R., Kelly, W., Chantelau, E., Gede, A., Sharman, D., O Halloran, D., Best, C., Abbas, Z. G., Lutale, J., Gill, G. V., Jarvis, W. R., Archibald, L. K., Corcoran, S., Mansell, J., Pibworth, L., Terada, H., Shiba, T., Utugi, N., Utugi, T., Blum, M., Strobel, J., Hoffken, K., Razvi, F. M., Kritzinger, E. E., Taylor, K., Jones, S., Illahi, W., Grubetaer, M., Hartmann, P., Hoffstadt, K., Leiden, H. A., Moll, A. C., Polak, B. C. P., Pietragalla, G. B., Maurino, M., Montanaro, M., Karadeniz, S., Tommasini, P., Quadrini, C., Demiraj, V., Rispoli, E., Ota, A., Takama, H., Saito, N., Hemandez, C., Lepore, D., Antico, L., Giardina, B., Franconi, F., Michoud, E., Chamot, S., Riva, Ch, Hammes, H-P, Renner, O., Breier, G., Lin, J., Alt, A., Betzholtz, C., Bretzel, R. G. Rd, Manti, R., Gallo, M., Molinar Hin, A., Brignardello, E., Boccuzzi, G., Li, Shanfang, Xiang, Kunsan, Zhang, Rugeng, Shangguan, Xinhong, Wu, Jianrong, Donnan, P. T., Broomhall, J., Hunter, K., Morris, A. D., Darts Memo, Collaboration, Ioannidis, G., Peppa, M., Rontogianni, E., Kallifronas, M., Lekatsas, I., Chrysanthopoulou, G., Anthopoulos, L., Kesse, M., Thalassinos, N., Neves, C., Medina, J. L., Lopes, F., Guvener, N., Guvener, M., Kocagoz, T., Boke, E., Pasaoglu, I., Bascil Tutuncu, N., Oto, A., Karvonen, M. K., Koulu, M., Pesonen, U., Mercuri, M., Rauramaa, R., Pittsburgh Epidemiology of Diabetes Complications (EDC) Study, Rutter, M. K., Kestevan, P., Mccomb, J. M., Marshall, S. M., Sobieska, M., Wiktorowicz, K., Kanters, S. D. J. M., Banga, J. D., Algra, A., Frijns, C. J. M., Beutler, J. J., Fijnheer, R., Nicoloff, G., Baydanoff, S., Stanimirova, N., Petrova, Ch, Lario, S., Campistol, J. M., Cases, A., Claria, J., Inigo, P., Esmatjcs, E., Sarman, B., Toth, M., Kocsis, I., Somogyi, A., Bumbure, A., Jachimowicz, K., Samson, J., Tomasiak, M., Sobol, A., Stanczyk, L., Watala, C., Stradina, P., Wisniewska-Jarosinska, M., Marciniak, D., Wieclawska, B., Golanski, J., Zinnat, R., Mahmud, I., Buyukasik, Yahya, Demiroglu, H., Szczepanik, A., Skowronski, M., Murawska, A., Meeking, D. R., Allard, S., Munday, J., Chowienczyk, P., Shaw, K. M., Cummings, M. H., Simkova, R., Jirsa, M., Hadoke, P. W. F., Mcintyre, C. A., Jones, G. C., Williams, B. C., Elliott, A. I., Mcknight, J. A., Pernow, J., Bombonato, G. C., Finucci, G. F., Zotta, L., Senses, V., Ozyazgan, S., Ince, E., Tuncdemir, M., Sultuybek, G., Akkan, A. G., Unlucerci, Y., Bekpinar, S., Meyer, M. F., Lee, B. C., Shore, A. C., Humphreys, J. M., Tooke, J. 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27. Regular exercise enhances insulin activation of IRS-1-associated PI3- kinase in human skeletal muscle
- Author
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Kirwan, J. P., Del Aguila, L. F., Jazmir Hernandez, Williamson, D. L., O Gorman, D. J., Lewis, R., and Krishnan, R. K.
28. The intranuclear cleft of the intervertebral disk: magnetic resonance imaging.
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Aguila, L A, primary, Piraino, D W, additional, Modic, M T, additional, Dudley, A W, additional, Duchesneau, P M, additional, and Weinstein, M A, additional
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- 1985
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29. Comparative analysis of Piezo-ICSI and conventional ICSI in bovine embryo development.
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Fuentes F, Aguila L, Pérez F, Muñoz E, Arias ME, and Felmer R
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- Animals, Cattle embryology, Male, Female, Embryo Culture Techniques veterinary, Embryo Culture Techniques methods, Spermatozoa physiology, Sperm Injections, Intracytoplasmic veterinary, Sperm Injections, Intracytoplasmic methods, Embryonic Development
- Abstract
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique (ART) mainly used to overcome severe male factor infertility problems in humans and animals. However, in cattle, one of the most demanded species for its meat and milk, the efficiency of this technique is low. The present study compared the effect of the piezoelectric and conventional injection systems on the preimplantational development and quality of bovine embryos generated by ICSI. Evaluations of the conditions for performing the Piezo-ICSI procedure showed that the application of a strong pulse (I4S7) was more effective in damaging the sperm plasma and acrosomal membranes prior to injection, compared to a soft pulse (I2S2, P < 0.05). In addition, Piezo-ICSI embryos without the application of exogenous activators achieved similar levels of development as Piezo-ICSI embryos activated with ionomycin and anisomycin (P > 0.05). When comparing conventional and piezoelectric injection systems, no significant differences in embryo development were observed (P > 0.05). However, embryos generated by Piezo-ICSI showed a higher embryo quality in terms of total cell number (P < 0.05). In addition, Piezo-ICSI embryos showed an expression profile of genes essential for embryonic development similar to IVF embryos (P > 0.05), in contrast to conventional ICSI-derived embryos, which presented overexpression of CASP3 and IFNT2 (P < 0.05). In conclusion, we confirmed that Piezo-ICSI is a more convenient approach than traditional ICSI, since does not require exogenous activation and generate embryos of better quality, regarding the total number of blastomeres and the pattern of gene expression observed., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2025
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30. Replacing sperm with genotyped haploid androgenetic blastomeres to generate cattle with predetermined paternal genomes†.
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Aguila L, Sampaio RV, Therrien J, Nociti RP, Labrecque R, Tremblay A, Marras G, Blondin P, and Smith LC
- Subjects
- Cattle genetics, Animals, Male, Female, Pregnancy, Genotype, Genome, Cloning, Organism methods, Cloning, Organism veterinary, Embryonic Development genetics, Sperm Injections, Intracytoplasmic, Blastomeres metabolism, Blastomeres cytology, Haploidy, Spermatozoa
- Abstract
Although meiosis plays an essential role for the survival of species in natural selection, the genetic diversity resulting from sexual reproduction impedes human-driven strategies to transmit the most suitable genomes for genetic improvement, forcing breeders to select diploid genomes generated after fertilization, that is, after the encounter of sperm and oocytes carrying unknown genomes. To determine whether genomic assessment could be used before fertilization, some androgenetic haploid morula-stage bovine embryos derived from individual sperm were biopsied for genomic evaluation and others used to reconstruct "semi-cloned" (SC) diploid zygotes by the intracytoplasmic injection into parthenogenetically activated oocytes, and the resulting embryos were transferred to surrogate females to obtain gestations. Compared to controls, in vitro development to the blastocyst stage was lower and fewer surrogates became pregnant from the transfer of SC embryos. However, fetometric measurements of organs and placental membranes of all SC conceptuses were similar to controls, suggesting a normal post-implantation development. Moreover, transcript amounts of imprinted genes IGF2, IGF2R, PHLDA2, SNRPN, and KCNQ1OT1 and methylation pattern of the KCNQ1 DMR were unaltered in SC conceptuses. Overall, this study shows that sperm can be replaced by genotyped haploid embryonic-derived cells to produce bovine embryos carrying a predetermined paternal genome and viable first trimester fetuses after transfer to female recipients., Summary Sentence: Haploid morula-stage embryonic cells derived from a single sperm can be genotyped and injected into activated oocytes to reconstruct diploid zygotes that develop both in vitro into blastocysts and in vivo into viable post-implantation bovine conceptuses with predetermined paternal genomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2024
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- View/download PDF
31. Production of bovine embryos by piezo-ICSI using capacitated spermatozoa selected by fluorescence-activated cell sorting (FACS-piezo-ICSI).
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Castro M, Aguila L, Arias ME, and Felmer R
- Subjects
- Animals, Male, Cattle embryology, Female, Heparin pharmacology, Sperm Capacitation drug effects, Flow Cytometry veterinary, Spermatozoa physiology, Spermatozoa drug effects, Sperm Injections, Intracytoplasmic veterinary, Sperm Injections, Intracytoplasmic methods
- Abstract
Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2 hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MβCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca
2+ ) levels and acrosome integrity were selected by FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species., Competing Interests: Declaration of Competing Interest The authors declare to have no conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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32. Haploid androgenetic development of bovine embryos reveals imbalanced WNT signaling and impaired cell fate differentiation†.
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Aguila L, Nociti RP, Sampaio RV, Therrien J, Meirelles FV, Felmer RN, and Smith LC
- Subjects
- Humans, Animals, Cattle, Haploidy, Cell Differentiation genetics, Blastocyst metabolism, Embryonic Development genetics, Wnt Signaling Pathway genetics, Gene Expression Regulation, Developmental
- Abstract
Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathways. To evaluate the role of WNT on hAE competence, we exposed early Day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental outcome of bovine hAE., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2023
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33. Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.
- Author
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Valencia C, Pérez-García F, Aguila L, Felmer R, and Arias ME
- Subjects
- Cattle, Animals, Mitogen-Activated Protein Kinases, Adenine pharmacology, Oocytes, Cycloheximide pharmacology, Blastocyst, Anisomycin pharmacology, Mammals, Parthenogenesis, Maturation-Promoting Factor
- Abstract
Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1
Thr161 , and p-CDK1Thr14-Tyr15 , were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations ( p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments ( p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment ( p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.- Published
- 2023
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34. Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR.
- Author
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Muñoz E, Castro M, Aguila L, Contreras MJ, Fuentes F, Arias ME, and Felmer R
- Subjects
- Horses, Male, Animals, Cattle, Female, Oocytes, Sperm Injections, Intracytoplasmic veterinary, Sperm Injections, Intracytoplasmic methods, Reference Standards, Semen, Spermatozoa
- Abstract
Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes.
- Published
- 2023
- Full Text
- View/download PDF
35. Effect of Atomized Black Maca ( Lepidium meyenii) Supplementation in the Cryopreservation of Alpaca ( Vicugna pacos) Epididymal Spermatozoa.
- Author
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Levano G, Quispe J, Vargas D, García M, López A, Aguila L, and Valdivia M
- Abstract
Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.
- Published
- 2023
- Full Text
- View/download PDF
36. Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos.
- Author
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Arias ME, Vargas T, Gallardo V, Aguila L, and Felmer R
- Abstract
Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showed similar in vitro competence compared to the undefined (FBS) control. In Experiment 4, completely defined conditions (absence of FBS and BSA during in vitro maturation and embryo culture) were tested. A higher cleavage was observed with FGF2 (86%) compared to EGF (77%) and the FBS control (77%), but similar blastocyst rates were observed for FGF2 (24%), EGF (19%) and the FBS control (25%). Embryo quality was similar among groups. Finally, post-thawing survival was higher for FGF2 (94%) compared to the FBS control (77%). Thus, we report a simple defined IVP system for bovine species that generates developmental outcomes and embryos of similar quality than those produced under conditions containing FBS.
- Published
- 2022
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- View/download PDF
37. Pluripotent Core in Bovine Embryos: A Review.
- Author
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Aguila L, Osycka-Salut C, Treulen F, and Felmer R
- Abstract
Early development in mammals is characterized by the ability of each cell to produce a complete organism plus the extraembryonic, or placental, cells, defined as pluripotency. During subsequent development, pluripotency is lost, and cells begin to differentiate to a particular cell fate. This review summarizes the current knowledge of pluripotency features of bovine embryos cultured in vitro, focusing on the core of pluripotency genes ( OCT4 , NANOG , SOX2 , and CDX2 ), and main chemical strategies for controlling pluripotent networks during early development. Finally, we discuss the applicability of manipulating pluripotency during the morula to blastocyst transition in cattle species.
- Published
- 2022
- Full Text
- View/download PDF
38. [Evaluation of a carbon 13 breath test for the diagnosis of Helicobacter pylori].
- Author
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Bernui G, Del Aguila L, Sanes M, Prochazka R, and Bussalleu A
- Subjects
- Adolescent, Adult, Breath Tests methods, Carbon Isotopes, Cross-Sectional Studies, Humans, Sensitivity and Specificity, Helicobacter Infections diagnosis, Helicobacter pylori
- Abstract
Objective: To evaluate a Chinese-made carbon-13 breath test as a non-invasive diagnostic method for Hp infection in the Peruvian population through the determination of sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio, and diagnostic utility., Materials and Methods: A cross-sectional and descriptive study was carried out on a secondary database of the Gastroenterology Service of the Cayetano Heredia Hospital and Cayetano Heredia Clinic from November 2017 to August 2018. Patients over 18 years of age who underwent endoscopy were included upper digestive., Results: A sensitivity of 90.3% (95% CI 0.81-0.96), a specificity of 82.5% (95% CI 0.67-0.93), a positive predictive value was found and negative of 90.3% and 82.5%, and positive and negative likelihood ratio of 5.16 and 0.12, respectively. The area under the ROC curve was 0.88 (95% CI: 0.80-0.96)., Conclusions: The carbon 13 breath test is a non-invasive and simple test that provides results quickly. However, it is not possible to recommend the routine use of this test due to the discrepancy between the international and local results obtained. Studies with larger sample sizes, standardized protocols and different manufacturers are suggested to evaluate their performance and characteristics.
- Published
- 2022
39. Core Histones Are Constituents of the Perinuclear Theca of Murid Spermatozoa: An Assessment of Their Synthesis and Assembly during Spermiogenesis and Function after Gametic Fusion.
- Author
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Hamilton LE, Lion M, Aguila L, Suzuki J, Acteau G, Protopapas N, Xu W, Sutovsky P, Baker M, and Oko R
- Subjects
- Animals, Cell Nucleus metabolism, Chromatography, Liquid methods, Female, Fertilization physiology, Male, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Sperm Injections, Intracytoplasmic, Tandem Mass Spectrometry methods, Histones biosynthesis, Sperm Head metabolism, Spermatids metabolism, Spermatogenesis physiology
- Abstract
The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo -synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo -synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.
- Published
- 2021
- Full Text
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40. Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos.
- Author
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Aguila L, Suzuki J, Hill ABT, García M, de Mattos K, Therrien J, and Smith LC
- Abstract
Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aguila, Suzuki, Hill, García, de Mattos, Therrien and Smith.)
- Published
- 2021
- Full Text
- View/download PDF
41. Oocyte Selection for In Vitro Embryo Production in Bovine Species: Noninvasive Approaches for New Challenges of Oocyte Competence.
- Author
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Aguila L, Treulen F, Therrien J, Felmer R, Valdivia M, and Smith LC
- Abstract
The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30-40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of noninvasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different noninvasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (>120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated with better competence. In addition, the combination of oocyte and zygote selection via brilliant cresyl blue (BCB) test, spindle imaging, and the anti-Stokes Raman scattering microscopy together with studies decoding molecular cues in oocyte maturation have the potential to further optimize the identification of oocytes with better developmental competence for in-vitro-derived technologies in livestock species.
- Published
- 2020
- Full Text
- View/download PDF
42. Effect of MnTBAP on in vitro capacitation of frozen-thawed stallion sperm.
- Author
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Treulen F, Aguila L, Arias ME, Jofré I, and Felmer R
- Subjects
- Animals, Male, Cryopreservation veterinary, Horses physiology, Metalloporphyrins pharmacology, Semen Preservation veterinary, Sperm Capacitation drug effects
- Abstract
In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 10
6 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating, and capacitating plus 150 mM MnTBAP. There were incubations for 4 h at 37.5 °C in a humidified air atmosphere. Sample analysis was performed immediately after thawing (0 h), and at the end of the incubation period (4 h), unless otherwise indicated. The following variables were evaluated for spermatozoa: plasma membrane integrity and fluidity, acrosome integrity, intracellular calcium concentrations, intracellular pH, tyrosine phosphorylation, ATP concentrations, motility and heterologous zona-binding assay, using flow cytometry, fluorescent microscopy and/or chemiluminescence, depending on the most appropriate procedure for the variable being evaluated. Results indicated that capacitation-like changes were synergistically induced by the cAMP agonists, phosphodiesterase inhibitor and bicarbonate. The presence of bovine serum albumin was harmful to the plasma membrane. The MnTBAP supplementation had a positive effect on viability-related markers (plasma membrane integrity, membrane fluidity, associated with greater intracellular pH) when there were capacitating conditions. In conclusion, the activity of MnTBAP contributes to improving the in vitro incubation conditions of frozen-thawed stallion sperm., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
43. Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies.
- Author
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Navarrete FA, Aguila L, Martin-Hidalgo D, Tourzani DA, Luque GM, Ardestani G, Garcia-Vazquez FA, Levin LR, Buck J, Darszon A, Buffone MG, Mager J, Fissore RA, Salicioni AM, Gervasi MG, and Visconti PE
- Abstract
To become fertile, mammalian sperm must undergo a series of biochemical and physiological changes known as capacitation. These changes involve crosstalk between metabolic and signaling pathways and can be recapitulated in vitro . In this work, sperm were incubated in the absence of exogenous nutrients (starved) until they were no longer able to move. Once immotile, energy substrates were added back to the media and sperm motility was rescued. Following rescue, a significantly higher percentage of starved sperm attained hyperactivated motility and displayed increased ability to fertilize in vitro when compared with sperm persistently incubated in standard capacitation media. Remarkably, the effects of this treatment continue beyond fertilization as starved and rescued sperm promoted higher rates of embryo development, and once transferred to pseudo-pregnant females, blastocysts derived from treated sperm produced significantly more pups. In addition, the starvation and rescue protocol increased fertilization and embryo development rates in sperm from a severely sub-fertile mouse model, and when combined with temporal increase in Ca
2+ ion levels, this methodology significantly improved fertilization and embryo development rates in sperm of sterile CatSper1 KO mice model. Intracytoplasmic sperm injection (ICSI) does not work in the agriculturally relevant bovine system. Here, we show that transient nutrient starvation of bovine sperm significantly enhanced ICSI success in this species. These data reveal that the conditions under which sperm are treated impact post-fertilization development and suggest that this "starvation and rescue method" can be used to improve assisted reproductive technologies (ARTs) in other mammalian species, including humans., (Copyright © 2019 Navarrete, Aguila, Martin-Hidalgo, Tourzani, Luque, Ardestani, Garcia-Vazquez, Levin, Buck, Darszon, Buffone, Mager, Fissore, Salicioni, Gervasi and Visconti.)- Published
- 2019
- Full Text
- View/download PDF
44. Sperm-borne glutathione-S-transferase omega 2 accelerates the nuclear decondensation of spermatozoa during fertilization in mice†.
- Author
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Hamilton LE, Suzuki J, Aguila L, Meinsohn MC, Smith OE, Protopapas N, Xu W, Sutovsky P, and Oko R
- Subjects
- Amino Acid Sequence, Animals, Female, Glutathione Transferase chemistry, Glutathione Transferase genetics, Male, Mice, Sperm Injections, Intracytoplasmic methods, Sperm-Ovum Interactions physiology, Glutathione Transferase metabolism, Sperm Head physiology, Spermatozoa enzymology
- Abstract
The postacrosomal sheath (PAS) of the perinuclear theca (PT) is the first compartment of the sperm head to solubilize into the ooplasm upon sperm-oocyte fusion, implicating its constituents in zygotic development. This study investigates the role of one such constituent, glutathione-S-transferase omega 2 (GSTO2), an oxidative-reductive enzyme found in the PAS and perforatorial regions of the PT. GSTO2 uses the conjugation of reduced glutathione, an electron donor shown to be compulsory in sperm disassembly within the ooplasm. The proximity of GSTO2 to the condensed sperm nucleus led us to hypothesize that this enzyme may facilitate nuclear decondensation by reducing disulfide bonds before the recruitment of GSTO enzymes from within the ooplasm. To test this hypothesis, we utilized a cell permeable isozyme-specific inhibitor, which fluoresces when bound to the active site of GSTO2, to functionally inhibit spermatozoa before performing intracytoplasmic sperm injections (ICSI) in mice. The technique allowed for targeted inhibition of solely PT-residing GSTO2, as all that is required for complete zygotic development is the injection of the mouse spermatozoon head. ICSI showed that inhibition of PT-anchored GSTO2 caused a delay in sperm nuclear decondensation, and further resulted in untimely embryo cleavage, and an increase in fragmentation beginning at the morula stage. The confounding effects of these developmental delays ultimately resulted in decreased blastocyst formation. This study implicates PT-anchored GSTO2 as an important facilitator of nuclear decondensation and reinforces the notion that the PAS-PT is a critical sperm compartment harboring molecules that facilitate zygotic development., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)
- Published
- 2019
- Full Text
- View/download PDF
45. Impact of post-thaw supplementation of semen extender with antioxidants on the quality and function variables of stallion spermatozoa.
- Author
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Treulen F, Aguila L, Arias ME, Jofré I, and Felmer R
- Subjects
- Animals, Cell Membrane, DNA Fragmentation, Lipid Peroxidation, Male, Membrane Potential, Mitochondrial, Reactive Oxygen Species, Semen Preservation methods, Antioxidants pharmacology, Cryopreservation veterinary, Horses physiology, Semen Analysis veterinary, Semen Preservation veterinary, Spermatozoa drug effects
- Abstract
During cryopreservation procedures, the spermatozoa are exposed to physical and chemical stressors that generate an increase in the intracellular concentration of reactive oxygen species (ROS). If ROS concentrations are too great, this can lead to a state of oxidative stress that are detrimental to sperm quality. The aim of this study was to ascertain the profile the ROS production and assess the effects of post-thaw supplementation of a semen extender with different antioxidant compounds on the quality and function variables of frozen-thawed stallion spermatozoa incubated in vitro. Frozen-thawed stallion spermatozoa (2 × 10
6 cells/mL) were incubated with three different antioxidants (MnTBAP, NAC and FeTPPS) for 4 h at 38 °C. An untreated sperm suspension and a fresh sample were included as controls. Plasma membrane integrity (SYBR-14/PI), intracellular ROS concentration (DHE and ROS-ID™ total ROS/Superoxide Detection Kit), lipid peroxidation (BODIPY), DNA damage (TUNEL) and mitochondrial membrane potential (ΔΨm; TMRE/SYTOX) were evaluated by flow cytometry and fluorescence microscopy. In addition, sperm motility was evaluated using the ISAS system. Evaluations were performed at 0 and 4 h of incubation. The results indicate that superoxide anion is the main ROS produced by frozen-thawed stallion spermatozoa and that the use of MnTBAP improved sperm motility and viability, decreased the lipid peroxidation and DNA damage. In conclusion, this study provides relevant data to improve in vitro incubations conditions and to establish futures therapies using MnTBAP after thawing with the aim being to overcome the deleterious effects of semen cryopreservation and consequently preserve the stallion sperm quality through avoiding oxidative stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
46. Cryopreservation induces mitochondrial permeability transition in a bovine sperm model.
- Author
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Treulen F, Arias ME, Aguila L, Uribe P, and Felmer R
- Subjects
- Animals, Cattle, Cell Death, Cell Membrane physiology, Flow Cytometry, Male, Permeability, Semen Analysis, Calcium metabolism, Cryopreservation methods, Membrane Potential, Mitochondrial physiology, Mitochondria pathology, Spermatozoa physiology
- Abstract
When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 10
6 mL-1 and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca2+ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality., (Copyright © 2018. Published by Elsevier Inc.)- Published
- 2018
- Full Text
- View/download PDF
47. [Cultural Adaptation of the Peruvian Version of the Muscle Appearance Satisfaction Scale (MASS) for Muscle Dysmorphia].
- Author
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Cook-Del Aguila L, Sanchez-Castro AE, Yacila GA, Reyes Bossio MA, and Mayta-Tristán P
- Subjects
- Adolescent, Adult, Body Dysmorphic Disorders psychology, Humans, Male, Peru, Young Adult, Body Dysmorphic Disorders diagnosis, Body Image psychology, Muscle, Skeletal, Psychiatric Status Rating Scales
- Published
- 2016
- Full Text
- View/download PDF
48. Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.
- Author
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Zambrano F, Aguila L, Arias ME, Sánchez R, and Felmer R
- Subjects
- Acrosome drug effects, Acrosome physiology, Acrosome Reaction drug effects, Animals, Cell Membrane drug effects, Cryopreservation veterinary, DNA Fragmentation drug effects, Female, In Situ Nick-End Labeling, Male, Semen Preservation veterinary, Spermatozoa physiology, Spermatozoa ultrastructure, Type C Phospholipases analysis, Type C Phospholipases metabolism, Cattle embryology, Embryonic Development, Glycoproteins pharmacology, Lysophospholipase pharmacology, Octoxynol pharmacology, Sperm Injections, Intracytoplasmic veterinary, Spermatozoa drug effects
- Abstract
In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
49. High-density grids for efficient data collection from multiple crystals.
- Author
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Baxter EL, Aguila L, Alonso-Mori R, Barnes CO, Bonagura CA, Brehmer W, Brunger AT, Calero G, Caradoc-Davies TT, Chatterjee R, Degrado WF, Fraser JS, Ibrahim M, Kern J, Kobilka BK, Kruse AC, Larsson KM, Lemke HT, Lyubimov AY, Manglik A, McPhillips SE, Norgren E, Pang SS, Soltis SM, Song J, Thomaston J, Tsai Y, Weis WI, Woldeyes RA, Yachandra V, Yano J, Zouni A, and Cohen AE
- Subjects
- Animals, Crystallization economics, Crystallization methods, Crystallography, X-Ray economics, Crystallography, X-Ray methods, Data Collection, Diffusion, Equipment Design, Humans, Temperature, Volatilization, Crystallization instrumentation, Crystallography, X-Ray instrumentation
- Abstract
Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.
- Published
- 2016
- Full Text
- View/download PDF
50. Goniometer-based femtosecond crystallography with X-ray free electron lasers.
- Author
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Cohen AE, Soltis SM, González A, Aguila L, Alonso-Mori R, Barnes CO, Baxter EL, Brehmer W, Brewster AS, Brunger AT, Calero G, Chang JF, Chollet M, Ehrensberger P, Eriksson TL, Feng Y, Hattne J, Hedman B, Hollenbeck M, Holton JM, Keable S, Kobilka BK, Kovaleva EG, Kruse AC, Lemke HT, Lin G, Lyubimov AY, Manglik A, Mathews II, McPhillips SE, Nelson S, Peters JW, Sauter NK, Smith CA, Song J, Stevenson HP, Tsai Y, Uervirojnangkoorn M, Vinetsky V, Wakatsuki S, Weis WI, Zadvornyy OA, Zeldin OB, Zhu D, and Hodgson KO
- Subjects
- Crystallization, Electrons, Lasers, Models, Molecular, Myoglobin chemistry, RNA Polymerase II chemistry, Receptors, Adrenergic, beta-2 chemistry, Reproducibility of Results, Synchrotrons, X-Ray Diffraction methods, X-Rays, Chemistry, Physical instrumentation, Crystallography, X-Ray methods, Protein Conformation, Proteins chemistry
- Abstract
The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.
- Published
- 2014
- Full Text
- View/download PDF
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