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12. The cytokine platelet factor 4 successfully replaces bovine serum albumin for the in vitro culture of porcine embryos

Author

Cambra, J. M., Martinez-Serrano, Cristina, Maside, C., Rodriguez-Martinez, Heriberto, Martinez, E. A., Gil, M. A., Cuello, C., Cambra, J. M., Martinez-Serrano, Cristina, Maside, C., Rodriguez-Martinez, Heriberto, Martinez, E. A., Gil, M. A., and Cuello, C.

Abstract

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100 -1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 +/- 10.9 to 70.0 +/- 5.8%); of day 7-blastocyst rates (range: 46.6 +/- 10.0 to 56.4 +/- 8.2%) and of total day 7-blastocyst efficiency (range: 32.3 +/- 8.3 to 37.2 +/- 7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 +/- 23.2 to 50.5 +/- 26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500 ng/mL (range: 29.9 +/- 7.8 to 31.8 +/- 5.5%; P < 0.05) compared with BSA-control (17.2 +/- 8.2%) and PF4 1000 ng/mL (15.5 +/- 7.9%); showing similar blastocyst rates (range: 42.0 +/- 11.5 to 49.3 +/- 10.0%), total efficiency (28.0 +/- 8.2 to 32.3 +/- 7.1%) total cell numbers (range: 42.6 +/- 19.3 to 45.7 +/- 23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions. (C) 2019 Elsevier Inc. All rights reserved., Funding Agencies|Ministry of Science, Innovation and Universities-ERDF, Madrid, Spain [RTI2018-093525-B-I00]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15]; Research Council FORMAS, Stockholm, Sweden [2017e00946]

Published
2020
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15. Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence

Author

Cadenas, J., primary, Maside, C., additional, Ferreira, A.C.A., additional, Vieira, L.A., additional, Leiva-Revilla, J., additional, Paes, V.M., additional, Alves, B.G., additional, Brandão, F.Z., additional, Rodrigues, A.P.R., additional, Wheeler, M.B., additional, and Figueiredo, J.R., additional

Published
2018
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18. Caprine ovarian follicle requirements differ between preantral and early antral stages after IVC in medium supplemented with GH and VEGF alone or in combination

Author

Cadenas, J., primary, Leiva-Revilla, J., additional, Vieira, L.A., additional, Apolloni, L.B., additional, Aguiar, F.L.N., additional, Alves, B.G., additional, Lobo, C.H., additional, Rodrigues, A.P.R., additional, Apgar, G.A., additional, Smitz, J., additional, Figueiredo, J.R., additional, and Maside, C., additional

Published
2017
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22. EMBRYOLOGY

Author

Furia, G. U., primary, Kostelijk, E. H., additional, Vergouw, C. G., additional, Lee, H., additional, Lee, S., additional, Park, D., additional, Kang, H., additional, Lim, C., additional, Yang, K., additional, Park, Y., additional, Shin, M., additional, Beyhan, Z., additional, Fisch, J. D., additional, Sher, G., additional, Keskintepe, L., additional, VerMilyea, M. D., additional, Anthony, J. T., additional, Graham, J. R., additional, Tucker, M. J., additional, Freour, T., additional, Lattes, S., additional, Lammers, J., additional, Mansour, W., additional, Jean, M., additional, Barriere, P., additional, El Danasouri, I., additional, Gagsteiger, F., additional, Rinaldi, L., additional, Selman, H., additional, Antonova, I., additional, Milachich, T., additional, Valkova, L., additional, Shterev, A., additional, Barcroft, J., additional, Dayoub, N., additional, Thong, J., additional, Abdel Reda, H., additional, Khalaf, Y., additional, El Touky, T., additional, Cabry, R., additional, Brzakowski, R., additional, Lourdel, E., additional, Brasseur, F., additional, Copin, H., additional, Merviel, P., additional, Yamada, M., additional, Takanashi, K., additional, Hamatani, T., additional, Akutsu, H., additional, Fukunaga, T., additional, Inoue, O., additional, Ogawa, S., additional, Sugawara, K., additional, Okumura, N., additional, Chikazawa, N., additional, Kuji, N., additional, Umezawa, A., additional, Tomita, M., additional, Yoshimura, Y., additional, Van der Jeught, M., additional, Ghimire, S., additional, O'Leary, T., additional, Lierman, S., additional, Deforce, D., additional, Chuva de Sousa Lopes, S., additional, Heindryckx, B., additional, De Sutter, P., additional, Herrero, J., additional, Tejera, A., additional, De los Santos, M. J., additional, Castello, D., additional, Romero, J. L., additional, Meseguer, M., additional, Leperlier, F., additional, Mirallie, S., additional, Schats, R., additional, Al-Nofal, M., additional, Lens, J. W., additional, Rooth, H., additional, Hompes, P. G., additional, Lambalk, C. B., additional, Hreinsson, J., additional, Karlstrom, P. O., additional, Wanggren, K., additional, Lundqvist, M., additional, Vahabi, Z., additional, Eftekhari-Yazdi, P., additional, Dalman, A., additional, Ebrahimi, B., additional, Daneshzadeh, M. T., additional, Rajabpour Niknam, M., additional, Choi, E. G., additional, Rho, Y. H., additional, Oh, D. S., additional, Park, L. S., additional, Cheon, H. S., additional, Lee, C. S., additional, Kong, I. K., additional, Lee, S. C., additional, Liebenthron, J., additional, Montag, M., additional, Koster, M., additional, Toth, B., additional, Reinsberg, J., additional, van der Ven, H., additional, Strowitzki, T., additional, Morita, H., additional, Hirosawa, T., additional, Watanabe, S., additional, Wada, T., additional, Kamihata, M., additional, Kuwahata, A., additional, Ochi, M., additional, Horiuchi, T., additional, Fatemeh, H., additional, Karimian, L., additional, Fazel, M., additional, Fouladi, H., additional, Johansson, L., additional, Ruttanajit, T., additional, Chanchamroen, S., additional, Sopaboon, P., additional, Seweewanlop, S., additional, Sawakwongpra, K., additional, Jindasri, P., additional, Jantanalapruek, T., additional, Charoonchip, K., additional, Vajta, G., additional, Quangkananurug, W., additional, Yi, G., additional, Jo, J. W., additional, Jee, B. C., additional, Suh, C. S., additional, Kim, S. H., additional, Zhang, Y., additional, Zhao, H. J., additional, Cui, Y. G., additional, Gao, C., additional, Gao, L. L., additional, Liu, J. Y., additional, Sozen, E., additional, Buluc, B., additional, Vicdan, K., additional, Akarsu, C., additional, Tuncay, G., additional, Hambiliki, F., additional, Bungum, M., additional, Agapitou, K., additional, Makrakis, E., additional, Liarmakopoulou, S., additional, Anagnostopoulou, C., additional, Moustakarias, T., additional, Giannaris, D., additional, Wang, J., additional, Andonov, M., additional, Linara, E., additional, Charleson, C., additional, Ahuja, K. K., additional, Ozsoy, S., additional, Morris, M. B., additional, Day, M. L., additional, Cobo, A., additional, Viloria, T., additional, Campos, P., additional, Vallejo, B., additional, Remohi, J., additional, Roldan, M., additional, Perez-Cano, I., additional, Cruz, M., additional, Martinez, M., additional, Gadea, B., additional, Munoz, M., additional, Garrido, N., additional, Mesut, N., additional, Ciray, H. N., additional, Mesut, A., additional, Isler, A., additional, Bahceci, M., additional, Fortuno, S., additional, Legidos, V., additional, Muela, L., additional, Galindo, N., additional, Gunasheela, S., additional, Gunasheela, D., additional, Ueno, S., additional, Uchiyama, K., additional, Kondo, M., additional, Ito, M., additional, Kato, K., additional, Takehara, Y., additional, Kato, O., additional, Edgar, D. H., additional, Krapez, J. A., additional, Bacer Kermavner, L., additional, Virant-Klun, I., additional, Pinter, B., additional, Tomazevic, T., additional, Vrtacnik-Bokal, E., additional, Lee, S. G., additional, Kang, S. M., additional, Lee, S. W., additional, Jeong, H. J., additional, Lee, Y. C., additional, Lim, J. H., additional, Bochev, I., additional, Kyurkchiev, S., additional, Wilding, M., additional, Coppola, G., additional, Di Matteo, L., additional, Dale, B., additional, Hormann-Kropfl, M., additional, Kastelic, D., additional, Schenk, M., additional, Fourati Ben Mustapha, S., additional, Khrouf, M., additional, Braham, M., additional, Kallel, L., additional, Elloumi, H., additional, Merdassi, G., additional, Chaker, A., additional, Ben Meftah, M., additional, Zhioua, F., additional, Zhioua, A., additional, Kocent, J., additional, Neri, Q. V., additional, Rosenwaks, Z., additional, Palermo, G. D., additional, Best, L., additional, Campbell, A., additional, Fishel, S., additional, Calimlioglu, N., additional, Sahin, G., additional, Akdogan, A., additional, Susamci, T., additional, Bilgin, M., additional, Goker, E. N. T., additional, Tavmergen, E., additional, Cantatore, C., additional, Ding, J., additional, Depalo, R., additional, Smith, G. D., additional, Kasapi, E., additional, Panagiotidis, Y., additional, Papatheodorou, A., additional, Goudakou, M., additional, Pasadaki, T., additional, Nikolettos, N., additional, Asimakopoulos, B., additional, Prapas, Y., additional, Soydan, E., additional, Gulebenzer, G., additional, Karatekelioglu, E., additional, Budak, E., additional, Pehlivan Budak, T., additional, Alegretti, J., additional, Cuzzi, J., additional, Negrao, P. M., additional, Moraes, M. P., additional, Bueno, M. B., additional, Serafini, P., additional, Motta, E. L. A., additional, Elaimi, A., additional, Harper, J. C., additional, Stecher, A., additional, Baborova, P., additional, Wirleitner, B., additional, Schwerda, D., additional, Vanderzwalmen, P., additional, Zech, N. H., additional, Stanic, P., additional, Hlavati, V., additional, Gelo, N., additional, Pavicic-Baldani, D., additional, Sprem-Goldstajn, M., additional, Radakovic, B., additional, Kasum, M., additional, Strelec, M., additional, Simunic, V., additional, Vrcic, H., additional, Khan, I., additional, Urich, M., additional, Abozaid, T., additional, Ullah, K., additional, Abuzeid, M., additional, Fakih, M., additional, Shamma, N., additional, Ayers, J., additional, Ashraf, M., additional, Milik, S., additional, Pirkevi, C., additional, Atayurt, Z., additional, Yazici, S., additional, Yelke, H., additional, Kahraman, S., additional, Dal Canto, M., additional, Coticchio, G., additional, Brambillasca, F., additional, Mignini Renzini, M., additional, Novara, P., additional, Maragno, L., additional, Karagouga, G., additional, De Ponti, E., additional, Fadini, R., additional, Resta, S., additional, Magli, M. C., additional, Cavallini, G., additional, Muzzonigro, F., additional, Ferraretti, A. P., additional, Gianaroli, L., additional, Barberi, M., additional, Orlando, G., additional, Sciajno, R., additional, Serrao, L., additional, Fava, L., additional, Preti, S., additional, Bonu, M. A., additional, Borini, A., additional, Varras, M., additional, Polonifi, A., additional, Mantzourani, M., additional, Mavrogianni, D., additional, Stefanidis, K., additional, Griva, T., additional, Bletsa, R., additional, Dinopoulou, V., additional, Drakakis, P., additional, Loutradis, D., additional, Hickman, C. F. L., additional, Duffy, S., additional, Bowman, N., additional, Gardner, K., additional, Sati, L., additional, Zeiss, C., additional, Demir, R., additional, McGrath, J., additional, Yildiz, S., additional, Unal, S., additional, Kumtepe, Y., additional, Aljaser, F., additional, Hernandez, J., additional, Tomlinson, M., additional, Campbell, B., additional, Fosas, N., additional, Redondo Ania, M., additional, Marina, F., additional, Molfino, F., additional, Martin, P., additional, Perez, N., additional, Carrasco, A., additional, Garcia, N., additional, Gonzalez, S., additional, Marina, S., additional, Scaruffi, P., additional, Stigliani, S., additional, Tonini, G. P., additional, Venturini, P. L., additional, Anserini, P., additional, Guglielmo, M. C., additional, Albertini, D. F., additional, Lain, M., additional, Caliari, I., additional, Oikonomou, Z., additional, Chatzimeletiou, K., additional, Sioga, A., additional, Oikonomou, L., additional, Kolibianakis, E., additional, Tarlatzis, B., additional, Nottola, S. A., additional, Bianchi, V., additional, Lorenzo, C., additional, Maione, M., additional, Macchiarelli, G., additional, Gomez, E., additional, Gil, M. A., additional, Sanchez-Osorio, J., additional, Maside, C., additional, Martinez, M. J., additional, Torres, I., additional, Rodenas, C., additional, Cuello, C., additional, Parrilla, I., additional, Molina, G., additional, Garcia, A., additional, Margineda, J., additional, Navarro, S., additional, Roca, J., additional, Martinez, E. A., additional, Avcil, F., additional, Ozden, H., additional, Candan, Z. N., additional, Uslu, H., additional, Karaman, Y., additional, Gioacchini, G., additional, Giorgini, E., additional, Carnevali, O., additional, Ferraris, P., additional, Vaccari, L., additional, Choe, S., additional, Tae, J., additional, Kim, C., additional, Lee, J., additional, Hwang, D., additional, Kim, K., additional, Suh, C., additional, Jee, B., additional, Catt, S. L., additional, Sorenson, H., additional, Vela, M., additional, Duric, V., additional, Chen, P., additional, Temple-Smith, P. D., additional, Pangestu, M., additional, Yoshimura, T., additional, Fukunaga, N., additional, Nagai, R., additional, Kitasaka, H., additional, Tamura, F., additional, Hasegawa, N., additional, Kato, M., additional, Nakayama, K., additional, Takeuchi, M., additional, Aoyagi, N., additional, Yasue, K., additional, Watanabe, H., additional, Asano, E., additional, Hashiba, Y., additional, Asada, Y., additional, Iwata, K., additional, Yumoto, K., additional, Mizoguchi, C., additional, Sargent, H., additional, Kai, Y., additional, Ueda, M., additional, Tsuchie, Y., additional, Imajo, A., additional, Iba, Y., additional, Mio, Y., additional, Els-Smit, C. L., additional, Botha, M. H., additional, Sousa, M., additional, Windt-De Beer, M., additional, Kruger, T. F., additional, Muller, N., additional, Magli, C., additional, Corani, G., additional, Giusti, A., additional, Castelletti, E., additional, Gambardella, L., additional, Seshadri, S., additional, Sunkara, S. K., additional, El-Toukhy, T., additional, Kishi, I., additional, Maruyama, T., additional, Ohishi, M., additional, Akiba, Y., additional, Asada, H., additional, Konishi, Y., additional, Nakano, M., additional, Kamei, K., additional, Lee, J. H., additional, Lee, K. H., additional, Park, I. H., additional, Sun, H. G., additional, Kim, S. G., additional, Kim, Y. Y., additional, Choi, E. M., additional, Lee, D. H., additional, Chavez, S. L., additional, Loewke, K. E., additional, Behr, B., additional, Han, J., additional, Moussavi, F., additional, Reijo Pera, R. A., additional, Yokota, H., additional, Yokota, Y., additional, Yokota, M., additional, Sato, S., additional, Nakagawa, M., additional, Sato, M., additional, Anazawa, I., additional, Araki, Y., additional, Knez, K., additional, Pozlep, B., additional, Vermilyea, M. D., additional, Levy, M. J., additional, Carvalho, M., additional, Cordeiro, I., additional, Leal, F., additional, Aguiar, A., additional, Nunes, J., additional, Rodrigues, C., additional, Soares, A. P., additional, Sousa, S., additional, Calhaz-Jorge, C., additional, Braga, D. P. A. F., additional, Setti, A. S., additional, Figueira, R. C. S., additional, Aoki, T., additional, Iaconelli, A., additional, Borges, E., additional, Ozkavukcu, S., additional, Sonmezer, M., additional, Atabekoglu, C., additional, Berker, B., additional, Ozmen, B., additional, Isbacar, S., additional, Ibis, E., additional, Menezes, J., additional, Lalitkumar, P. G. L., additional, Borg, P., additional, Ekwurtzel, E., additional, Nordqvist, S., additional, Vaegter, K., additional, Tristen, C., additional, Sjoblom, P., additional, Azevedo, M. C., additional, Remohi Gimenez, J., additional, Gamiz, P., additional, Albert, C., additional, Ferreira, R. C., additional, Resende, S., additional, Colturato, S. S., additional, Ferrer Buitrago, M., additional, Ferrer Robles, E., additional, Munoz Soriano, P., additional, Ruiz-Jorro, M., additional, Calatayud Lliso, C., additional, Rawe, V. Y., additional, Hanrieder, J., additional, Gulen-Yaldir, F., additional, Bergquist, J., additional, Stavreus-Evers, A., additional, Grunskis, A., additional, Bazarova, A., additional, Dundure, I., additional, Fodina, V., additional, Brikune, J., additional, Lakutins, J., additional, Pribenszky, C., additional, Cornea, M., additional, Reichart, A., additional, Uhereczky, G., additional, Losonczy, E., additional, Ficsor, L., additional, Lang, Z., additional, Ohgi, S., additional, Nakamura, C., additional, Hagiwara, C., additional, Kawashima, M., additional, Yanaihara, A., additional, Jones, G. M., additional, Biba, M., additional, Kokkali, G., additional, Vaxevanoglou, T., additional, Chronopoulou, M., additional, Petroutsou, K., additional, Sfakianoudis, K., additional, Pantos, K., additional, Romano, S., additional, Albricci, L., additional, Stoppa, M., additional, Cerza, C., additional, Sanges, F., additional, Fusco, S., additional, Capalbo, A., additional, Maggiulli, R., additional, Ubaldi, F., additional, Rienzi, L., additional, Ulrick, J., additional, Kilani, S., additional, Chapman, M., additional, Losada, C., additional, Ortega, I., additional, Pacheco, A., additional, Bronet, F., additional, Aguilar, J., additional, Ojeda, M., additional, Taboas, E., additional, Perez, M., additional, Munoz, E., additional, Pellicer, A., additional, Boumela, I., additional, Assou, S., additional, Haouzi, D., additional, Monzo, C., additional, Dechaud, H., additional, Hamamah, S., additional, Nakaoka, Y., additional, Hashimoto, S., additional, Amo, A., additional, Yamagata, K., additional, Nakano, T., additional, Akamatsu, Y., additional, Mezawa, T., additional, Ohnishi, Y., additional, Himeno, T., additional, Inoue, T., additional, Ito, K., additional, and Morimoto, Y., additional

Published
2012
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25. Automated Turbidimetry of Serum Lipoprotein(a)

Author

Borque, L., Maside, C., and Iglesias, A.

Subjects
540 Chemie, ddc:540
Abstract

We describe a simple iminunoturbidimetric method for quantifying lipoprotein(a) in serum based on latex-enhanced particle agglutination technology. Carboxylated latex particles (diameter 240 nm) covalently coated with F(ab')2 fragments of anti-lipoprotein(a) antibodies are incubated with the sample for 5 min at 37°C, and the resulting agglutination is quantified by measuring the change of turbidity produced at 700 nm. The assay is rapid, precise and fully automated on the Hitachi 911 analyser. The assay range is about 0.03—0.9 g/l. Average analytical recovery was 97.8%. Precision (CV) ranged from 1.9 to 3.1% at different lipoprotein(a) values. There was no interference from bilirubin, Intfalipid®, haemoglobin, plasminogen or apolipoprotein B. Comparisons with a latex nephelometric assay carried out on the Behring nephelometer analyser, and with three commercially available methods, a radioimmunoassay and two ELISA assays, gave good correlations (r > 0.95), although a large among-method variation in lipoprotein(a) values was found. We conclude that the proposed latex turbidimetric immunoassay method is suitable for routine use in clinical laboratories.

Published
1993

46. 90 OPEN PULLED STRAW VITRIFICATION OF IN VITROPORCINE BLASTOCYTS IN A CHEMICALLY DEFINED MEDIUM.

Author

Sanchez-Osorio, J., Cuello, C., Gomis, J., Maside, C., Gil, M. A., Parrilla, I., Roca, J., Vazquez, J. M., and Martinez, E. A.

Subjects
CRYOPRESERVATION of organs, tissues, etc., BLASTOMERES, FROZEN semen, ARTIFICIAL insemination of swine, BLASTOCYST, SULFOXIDES, THERIOGENOLOGY, POLYOXYMETHYLENE
Abstract

The aim of this study was to design a chemically defined medium for the vitrification of in vitroproduced porcine blastocysts avoiding the use of serum or serum components. Cumulus–oocyte complexes were matured in vitroin NCSU-23 for 44h and were inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16h to assess in vitrofertilization (IVF) parameters (N=200) or for 6 days (N=600) in order to obtain blastocysts. For chemically delipidation, 10μM forskolin was added to the culture medium on Day 5 of in vitroculture. On Day 2, embryos were evaluated for cleavage rate. On Day 6, embryos were assessed for blastocyst formation; only those blastocysts showing excellent morphological appearance were selected for vitrification. Blastocysts were vitrified using as basic medium TCM-199 HEPES supplemented with 20% of newborn calf serum (NBCS; n=65), with 0.1% of polyvinyl alcohol (PVA; n=64) or without additives (WA; n=65). The OPS-vitrification and warming were performed as described by (Sanchez-Osorio et al.2010 Theriogenology 73, 300–308) using 16% of Etylenglycol and 16% of dimetyl sulfoxide as final concentrations of cryoprotectants. Vitrified blastocysts were warmed and cultured in vitrofor 24h to assess their viability. Blastocysts that totally reformed their blastocoel cavity showing a normal or excellent morphology were considered viable. In addition, after in vitroculture vitrified-warmed viable embryos were fixed in 4% paraformaldehyde in PBS medium and stained with Hoechst 33342 in order to assess the total number of cells. Data were analysed by using the MIXED procedure of SPSS. The threshold for significance was set at P<0.05. Results are expressed as least squares means±SEM. The maturation, penetration, and monospermy rates were 98.5±1.2%, 85.3±3.6%, and 48.8±5%, respectively. The efficiency of IVF (defined as the ratio of monospermic oocytes to the total number of inseminated oocytes) was 41.0±4.9%. The values of cleavage rate at Day 2 and blastocysts formation rate were 67.8±1.4% and 37.3±1.6%, respectively. After vitrification and warming, similar survival rates were observed for NBCS (33.8±5.9) PVA (40.6±6.0), and WA (30.8±5.9) groups. No significant differences were found for the total number of cells (ranged from 35.4±6.8 to 50.8±8.3) among vitrification groups. In conclusion, in vitroderived porcine blastocysts can be vitrified in the absence of serum and serum components. Furthermore, PVA is a suitable substitute for serum in vitrification solutions with no detrimental effect on the viability of in vitroproduced pig blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07). [ABSTRACT FROM AUTHOR]

Published
2011
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47. 335 EFFECT OF MEM VITAMINS AND FORSKOLIN ON IN VITROEMBRYO PRODUCTION AND SOPS-VITRIFICATION ABILITY OF IN VITRODERIVED PORCINE BLASTOCYSTS.

Author

Cuello, C., Almiñana, C., Gomis, J., Gil, M. A., Maside, C., Gomez, S., Sanchez-Osorio, J., Roca, J., Vazquez, J. M., and Martinez, E. A.

Subjects
VITAMINS, FORSKOLIN, FERTILIZATION in vitro, BLASTOCYST, OVUM, ZYGOTES, ARTIFICIAL insemination of swine, SWINE, MAMMAL reproduction
Abstract

The aims of this study were, first to investigate the effect of minimum essential medium (MEM) vitamins (VITs) during IVM of porcine oocytes on in vitroembryo production of porcine embryos and second, to determine if the addition of VITs during IVM and the chemical delipidation with forskolin improve the vitrification ability of in vitro-derived porcine blastocysts. COCs were divided in two groups and matured in NCSU-23 for 44 h with 0.05% VITs (V group) or without VITs (NV group). Matured oocytes were inseminated with frozen-thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess IVF parameters (N= 767) or for 6 days (N= 2858) to evaluate the in vitroembryo development (Day 0 = day of IVF). On Day 5, some embryos from NV and V groups were cultured for 24 h with 10 μM forskolin (NVF and VF groups). The remaining embryos were cultured without forskolin (NVNF and VNF groups). On Day 6, embryos from the four experimental groups were assessed for blastocysts formation and some blastocysts (N= 414) were vitrified using superfine open pulled straws (SOPS). Vitrified blastocysts were warmed (one-step dilution method) and cultured for 24 h to assess their viability. Blastocysts with totally or partially reformed blastocoel cavity and normal or excellent morphology were considered viable. Five replicates of a 2 × 2 factorial design were conducted. Data was analyzed with the MIXED procedure. The threshold for significance was set at P< 0.05. Results are expressed as least squares means ± SEM. No differences were observed in the maturation of COCs treated with VITs (92.7 ± 1.3%) and non-treated COCs (94.3 ± 1.3%). The NV and V maturation groups showed similar penetration (83.0 ± 3% and 82.6 ± 3%, respectively) and monospermy (48.9 ± 6%, and 48.3 ± 6%, respectively) rates. The rate of monosermic oocytes related to the total of analyzed oocytes was similar for NV (38.1 ± 3.7%) and V (38.4 ± 3.7%) groups. The values of cleavage rate on Day 2 were similar for NV (66.7 ± 1.5%) and V (69.6 ± 1.6%) embryos. The addition of VITs to IVM medium improved (P< 0.01) up to 10 points the blastocysts formation rate, but the addition of forskolin at Day 5 did not affect this parameter. The V group showed a higher (P< 0.01) blastocysts rate (45.1 ± 2.5%) than the NV group (38.4 ± 2.3%).The addition of VITs did not affect the survival of in vitro-derived blastocysts after SOPS-vitrification on Day 6. However, the blastocysts cultured for 24 h with forskolin showed higher (P< 0.05) viability (44.0 ± 7.9%) after vitrification and warming than those embryos cultured without forskolin (34.1 ± 7.8%). In conclusion, the addition of MEM vitamins to IVM medium improves the blastocysts formation rate and the chemical delipidation with forskolin improve the cryosurvival of SOPS-vitrified porcine in vitro-derived blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07), MICINN (AGL2009-12091 and RC-2007), and CARM (2I05SU0012). [ABSTRACT FROM AUTHOR]

Published
2010
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48. Heat shock protein 70 kDa (HSP70) is involved in the maintenance of pig sperm function throughout liquid storage at 17 °C.

Author

Garriga F, Maside C, Padilla L, Recuero S, Rodríguez-Gil JE, and Yeste M

Subjects
Animals, Male, Swine, Mitochondria metabolism, Semen Analysis, Cell Survival drug effects, Calcium metabolism, HSP70 Heat-Shock Proteins metabolism, Spermatozoa metabolism, Spermatozoa physiology, Semen Preservation methods, Semen Preservation veterinary, Sperm Motility drug effects, Reactive Oxygen Species metabolism
Abstract

At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of cell lifespan. In this context, the study of proteins that could protect sperm during liquid storage is of high relevance. The 70 kDa Heat Shock Protein (HSP70) is an anti-apoptotic protein that has been reported to be relevant to sperm survival. Thus, we explored the role of HSP70 during prolonged storage of pig semen at 17 °C. Six semen pools were incubated with YM-1 (0, 0.05, 0.1 and 0.2 μM), an HSP70 inhibitor, and stored at 17 °C for 21 days. On days 0, 4, 10, 14 and 21, sperm quality and function were evaluated through flow cytometry and Computer-Assisted Sperm Analysis (CASA), and HSP70 activity and chromatin condensation were also determined. While inhibition of HSP70 increased progressive motility, Ca 2+ and Reactive Oxygen Species (ROS) levels, and mitochondrial activity during the first 10 days of storage, it had a detrimental effect on sperm motility after 14 and 21 days. In spite of this, sperm viability was not altered. We can conclude that HSP70 contributes to the liquid storage of pig semen because it keeps mitochondrial activity low, which is needed for the maintenance of sperm function., (© 2024. The Author(s).)

Published
2024
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49. Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Author

Maside C, Recuero S, Salas-Huetos A, Ribas-Maynou J, and Yeste M

Subjects
Male, Animals, Swine, Farms, Sperm Motility, Semen, Chromatin metabolism, DNA, Spermatozoa metabolism, Semen Analysis veterinary, Laboratories
Abstract

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Published
2023
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50. The effects of red LED light on pig sperm function rely upon mitochondrial electron chain activity rather than on a PKC-mediated mechanism.

Author

Blanco-Prieto O, Maside C, Àlex Peña, Ibáñez-Príncep J, Bonet S, Yeste M, and Rodríguez-Gil JE

Abstract

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20-28 ® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant ( p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 staining and O 2 consumption rate. Antimycin A, however, did not affect intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract the effects of light on motility, O 2 consumption rate and CCO activity, but not to the same extent than that observed for antimycin A. Finally, the effects observed when sperm were co-incubated with antimycin A and PKCi were similar to those observed with antimycin A alone. In conclusion, red LED light acts on sperm function via a direct effect on mitochondrial electron chain. Additionally, light-activated PKC pathways have a supplementary effect to that observed in the electron chain, thereby modulating sperm parameters such as motility and CCO activity., Competing Interests: JR-G, SB and MY are inventors of a patent entitled “Method and apparatus improving the quality of mammalian sperm” (European Patent Office No. 16199093.2; EP-3-323-289-A1), which is owned by Intruments Utils de Laboratori Geniul, S.L. (Barcelona, Spain) and an indirect relationship with the described experiments. The remaining 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 © 2022 Blanco-Prieto, Maside, Àlex Peña, Ibáñez-Príncep, Bonet, Yeste and Rodríguez-Gil.)

Published
2022
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