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Objective: The aim of the present study was to determine the effect of supplementing pasture-finished steers with corn silage on the expression level of the calpain system proteins and beef tenderization. Methods: Thirty Braford steers grazing on summer pasture were used for the study. For 120 days fifteen animals were supplemented with corn silage at 1% of body weight per head per day (Suppl) whereas the remaining 15 steers only received pasture (Contr). Carcass and meat traits were evaluated and compared between groups. Gene expression and activities of proteases (calpain 1 and calpain 2) and inhibitor (calpastatin) were measured using real-time polymerase chain reaction and casein zymography. Results: Carcass and meat traits were significantly different between feeding systems. Supplemented steers showed higher hot carcass weight (p

Objective: The aim of this study was to evaluate the effect of ageing and feeding strategies on the calpain protease system and meat quality traits in Braford steers. Methods: Thirty Braford steers were employed; 15 animals were supplemented with corn silage during finishing and 15 were kept only on pasture. Meat quality traits and calpain system protein activity were evaluated in longissimus thoracis et lumborum (LTL) steaks aged for 2, 7, 14, and 21 days. Results: Aged meat showed higher pH and calcium content, while Warner Bratzler shear force (WBSF) decreased to day 21. No interaction between ageing and diet was seen for quality traits. Steers finished with corn silage showed higher values of water holding capacity, WBSF and free calcium, and lower values of pH and cooking loss. Calpain and calpastatin activities decreased with ageing. Finishing steers on pasture produced higher values of calpains and lower values of calpastatin activities. The higher values of calpain 1 activity were observed in muscles aged 2 days from pasture finished animals, and the lower activity of the inhibitor in the 21 days aged samples of the same group. Conclusion: These results suggest a diet by ageing interaction in calpains and calpastatin and this interaction impact in Warner Bratzler Shear Force in Braford LTL muscle. Resumen: Objetivo: El objetivo de este estudio fue evaluar el efecto del envejecimiento y las estrategias de alimentación sobre el sistema de calpaína proteasa y los rasgos de calidad de la carne en novillos Braford. Métodos: Se emplearon 30 novillos Braford; 15 animales fueron suplementados con maíz ensilaje durante el acabado y 15 se mantuvieron solo en pasto. Características de la calidad de la carne y calpaína Se evaluó la actividad de la proteína del sistema en filetes longissimus thoracis et lumborum (LTL) envejecido durante 2, 7, 14 y 21 días. Resultados: La carne añejada mostró mayor pH y contenido de calcio, mientras que la carne cortada Warner Bratzler (WBSF) disminuyó hasta el día 21. No se observó interacción entre el envejecimiento y la dieta para rasgos de calidad. Los novillos terminados con ensilaje de maíz presentaron mayores valores de capacidad de retención de agua, WBSF y calcio libre, y menores valores de pH y mermas por cocción. Calpaína y calpastatina actividades disminuyeron con el envejecimiento. El engorde de novillos en pasto produjo valores más altos de calpaínas y valores más bajos de actividades de calpastatina. Los valores más altos de actividad de calpaína 1 se observaron en músculos de 2 días de edad de animales terminados en pasto, y la menor actividad del inhibidor en las muestras de 21 días de edad del mismo grupo. Conclusión: Estos resultados sugieren una interacción dieta por envejecimiento en calpaínas y calpastatina y esta interacción impacta en Warner Bratzler Shear Force en el músculo Braford LTL. Instituto de Investigación de Tecnología de Alimentos (ITA) Fil: Coria, María Sumampa. Instituto de Bionanotecnología del NOA (INBIONATEC). Laboratorio de Producción y Reproducción Animal; Argentina. Fil: Coria, María Sumampa. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustria. Instituto de Desarrollo Agropecuario del Semiárido (INDEAS); Argentina. Fil: Coria, María Sumampa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fil: Pighin, Darío Gabriel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación Tecnología de Alimentos; Argentina. Fil: Pighin, Darío Gabriel. Instituto de Ciencia y Tecnología de los Sistemas Alimentarios Sustentables (ICyTeSAS) UEDD INTA-CONICET; Argentina. Fil: Pighin, Darío Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina; Argentina. Fil: Pighin, Darío Gabriel. Universidad de Morón. Facultad de Agronomía y Ciencias Agroalimentarias; Argentina. Fil: Grigioni, Gabriela María. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación Tecnología de Alimentos; Argentina. Fil: Grigioni, Gabriela María. Instituto de Ciencia y Tecnología de Sistemas Alimentarios Sustentables (ICyTeSAS). UEDD INTA CONICET; Argentina. Fil: Grigioni, Gabriela María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fil: Grigioni, Gabriela María. Universidad de Morón. Facultad de Agronomía y Ciencias Agroalimentarias; Argentina. Fil: Palma, Gustavo Adolfo. Instituto de Bionanotecnología del NOA (INBIONATEC). Laboratorio de Producción y Reproducción Animal; Argentina. Fil: Palma, Gustavo Adolfo. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustria. Instituto de Desarrollo Agropecuario del Semiárido (INDEAS); Argentina. Fil: Palma, Gustavo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.

Objective. The aim of the present study was to determine the effect of supplementing pasturefinished Braford steers with corn silage on the expression level of genes associated with intramuscular fat in longissimus dorsi muscle. Materials and methods. Thirty Braford steers grazing on summer pasture were used for the study. For 120 days fifteen animals were supplemented with corn silage at 1% of body weight per head per day (Supl) whereas the remaining 15 steers only received pasture (Cont). Animals were slaughtered at 26 month of age with 464±17 Kg. Gene expression of glucose transporter type 4 (glut4), insulin-like growth factor 1 (igf1) and myostatin were measured using real-time polymerase chain reaction (RT-qPCR). Results. Supplementation produced increased expression of glut4 and igf1 genes. The expression of the genes studied was correlated with hot carcass weight, fat score of carcass and intramuscular fat content. Conclusions. Results suggest a gene expression-diet interaction in glut4 and igf1 genes, which impact on carcass fattening and intramuscular fat content in longissimus dorsi muscle of Braford steers, suggesting that these meat variables could be modulated through differential gene expression., Objetivo. Determinar el efecto de la suplementación con silo de maíz en la expresión de genes asociados al contenido de grasa intramuscular en el músculo longissimus dorsi de novillos Braford. Materiales y Métodos. Se utilizaron 30 novillos Braford. Durante 120 días 15 animales fueron suplementados con silo de maíz al 1% de su peso vivo (Supl), y 15 animales fueron alimentados solamente en pastura tropical (Cont). La faena se realizó a los 26 meses de edad con 464±17 Kg. Mediante la reacción en cadena de la polimerasa en tiempo real (RT-qPCR), se determinó la expresión de los genes transportador de glucosa 4 (glut4), factor de crecimiento tipo insulínico 1 (igf1) y miostatina. La expresión de genes fue asociada con parámetros de calidad de la canal y de la carne. Resultados. La suplementación con silo de maíz produjo mayor expresión de los genes glut4 e igf1. La expresión de los genes estudiados fue correlacionada con el peso de la carcasa caliente, el engrasamiento de la canal y el contenido de grasa de la carne. Conclusiones. Estos resultados sugieren interacción expresión genética-dieta en los genes glut4 y igf1, la cual impactan en el engrasamiento de la canal y el contenido de grasa intramuscular en músculo longissimus dorsi de los novillos Braford, sugiriendo que estas variables podrían ser moduladas a través de la expresión diferencial de los genes.

The objectives of the present study were (experiment 1) to characterized development and dynamics of the dominant follicles (DF) and the corpus luteum (CL) to determine patterns of two (W2) and three (W3) follicular waves in beef heifers, and (experiment 2) to determine gene expression of growth factors gene expression in follicular cells of W2 and W3 heifer. Twenty-eight Braford heifers were used. Dominant follicular and CL were monitored daily by ultrasonography to identify the development W2 and W3 in heifers. Pre-ovulatory DF were aspirated on day 19 in W2 and on day 22 in W3 heifers. In W2 and W3, follicular cells (FC) of gene expression of growth differentiation factor 9, bone morphogenetic protein 15 (BMP15), fibroblast growth factor basic, transforming growth factor beta receptor 1, bone morphogenetic protein receptor type IB and fibroblast growth factor receptor 2 were evaluated. The regression of the DF of the first follicular wave and the emergency of the DF of the second follicular wave began later in the heifers W2 than in W3 (p = .02 and p .01). The regression of the CL began earlier in the W2 than in W3 group (p .01). Gene expression of growth factors and receptors was similar between groups. However, higher relative levels of BMP15 was observed in W2 group (p = .07). Results propose that wave patterns were regulated by the development time of the DF in the first wave and the life of the CL. Furthermore, higher levels of BMP15 could produce shorter life of CL. The present work suggest that ultrasonography associated with molecular assays could be used as an easy and effective tool to characterize follicular wave patterns.

The objective of the present work was to evaluate the effect of J-Synch protocol on pregnancy rate (PR) of heifers artificially inseminated after detected estrus (DEAI) or at fixed-time (FTAI). Two hundred ninety-three Braford heifers were used. On day 0, all animals received 2.0 mg of estradiol benzoate administered intramuscularly (IM) and an intravaginal progesterone-releasing device (DIB, 0.5 g). Heifers were assigned to the following group: 1) J-Synch: on day 6, DIB devices were removed and 500 µg of d-Cloprostenol sodium (d-CS) was administered IM and paint was applied on tail base (tail-paint). On day 9 (60 h after DIB removal), heifers with ? 40% of the tail-paint rubbed off were inseminated. Those not showing estrus by 72 h after DIB removal received 100 µg of GnRH at that time and were FTAI. 2) Conventional: on day 7, DIB devices were removed, and 500 µg of d-CS and 0.5 mg of estradiol cypionate were administered IM and tail-paint. Forty-eight hours after DIB removal, heifers with the tail-paint rubbed off were DEAI. Those not showing estrus by 54 h after DIB removal were FTAI. Pregnancy rate was diagnosed by ultrasonography 30 days after FTAI in all heifers and did not differ between protocols (44.28% and 42.48%, P = 0.673). In both protocols, higher PR was observed in DEAI heifers (P = 0.047). In conclusion, the use of the J-Synch protocol generated similar PR to the Conventional protocol, allowing its use with the same efficiency. Furthermore, the DEAI generated better PR than the FTAI, regardless of the protocol used.

Introduction. Growth and follicular maturation involve transformations of various components of the follicle, such as the oocyte, granulosa and techa cells. Several growth factors, including differentiation growth factor 9 (GDF9), bone morphogenic protein 15 (BMP15) and basic fibroblast growth factor (FGF2) are important for follicular development and oocyte maturation, by its ability to increase the proliferation of granulosa, techa cells and the ovarian stroma. Objetive. Evaluate mRNA expression of GDF9, BMP15, FGF2 and their main receptors, transforming growth factor beta receptor 1 (TGFβ-R1), bone morphogenetic protein receptor, type IB (BMPR-IB) and fibroblast growth factor receptor 2 (FGFR2) in bovine follicular cells. Materials and methods. Total RNA was isolated from pooled samples of oocytes (OOs), cumulus cells (CCs) of cumulus oocyte complexes (COCs) and follicular cell pellets (PCs) of 70 ovaries obtained from 96 beef heifers, collected at a local abattoir. The expression pattern of growth factors and their receptors in follicular bovine cells was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Results. The mRNA transcripts encoding GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB and FGFR2 genes were detected, by RT-PCR, in all studied cells. This is the first time that the expression of TGFβ-R1 and BMPR-IB receptors is reported in bovine oocytes. Conclusions. The presence of growth factors and receptor transcripts in the studied cells indicate that these factors could act as paracrine and autocrine regulators of folliculogenesis., Introducción. El crecimiento y la maduración folicular implican una serie de transformaciones de diversos componentes del folículo, como el ovocito, las células de la granulosa y de la teca. Varios factores de crecimiento, incluyendo factor de crecimiento de diferenciación 9 (GDF9), proteína morfogénica ósea 15 (BMP15) y factor de crecimiento de fibroblastos básico (FGF2) son importantes para el desarrollo folicular y maduración de ovocitos, por su capacidad de aumentar la proliferación de las células de la granulosa, teca y el estroma ovárico. Objetivo. Evaluar la expresión de los factores de crecimiento GDF9, BMP15, FGF2 y sus principales receptores: el receptor 1 del factor de crecimiento transformante beta (TGFβ-R1), el receptor de la proteína morfogénica del hueso tipo IB (BMPR-IB) y el receptor del factor de crecimiento fibroblástico 2 (FGFR2) en células foliculares bovinas. Materiales y métodos. Se realizó la extracción de ARN total de pooles de ovocitos (OOs) y células del cumulus (CCs) de complejos cumulus-ovocito (COCs) y del pellet de células foliculares (PCs) provenientes de 70 ovarios obtenidos de 96 vaquillonas para carne, colectados en un frigorífico local. Los patrones de expresión de los factores de crecimiento y sus receptores en las células foliculares bovinas fueron evaluados por retro-transcripción seguida de la reacción en cadena de la polimerasa (RT-PCR). Resultados. La presencia de los transcriptos de ARNm de los genes GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB y FGFR2 fue detectada por RT-PCR en todas las células estudiadas. Es la primera vez que se reporta en ovocitos bovinos la expresión de los receptores TGFβ-R1 y BMPR-IB. Conclusiones. La presencia de transcriptos de factores de crecimiento y sus receptores en las células estudiadas, indica que estos factores podrían actuar como reguladores paracrinos y autocrinos de la foliculogénesis.

The objectives of this study were 1) to determine the effect of monensin treatment, alone or combined with a hormonal estrus synchronization treatment, on the pregnancy rate of lactating beef cows, and 2) to evaluate the effect of monensin capsule administration on ruminal metabolism in steers. In experiment 1, ninety-four cows were selected from a 300 cow herd. The experimental design used was a 2 x 2 factorial with the administration of monensin capsule as first factor (M1 = with monensin vs. M0 = without monensin) and hormonal treatment as second factor (H1 = with hormonal treatment vs. H0 = no hormonal treatment). Thirty-eight days before the beginning of the breeding season, cows were randomly assigned to the first factor, and thirty days later to the second factor, resulting in four treatments: M1H0, M1H1, M0H0 and M0H1. Cow were exposed to bull (bull/cow ratio 1:20) from day 0 (day 0 = start of the breeding season and 38 days after monensin capsule administration) to day 50. Pregnancy diagnosis was performed at 30, 60 and 80 days after start breeding season by ultrasonography. In experiment 2, eight ruminally cannulated crossbred beef steers were randomly assigned to two treatments (M1 and M0). To determine proportion of volatile fatty acids (VFA), ruminal fluid samples were taken on days 0, 40 and 77 of the experimental period, at 0, 4 and 12 h after grazing. In experiment 1, treatments whit monensin did not improve pregnancy rate (P = 0.95), however, hormonal treatment resulted in grater pregnancy rates (P = 0.03). In experiment 2, the proportion of VFA in ruminal fluid of steers was significantly different between treatments. The highest proportion of propionate was found in ruminal fluid from M1 treatment at 12 h after grazing (P = 0.04). In conclusion, the treatment with monensin increased the proportion of propionate. The result might suggest that energy balance was improved in steers, without improvement in cow´s pregnancy rates. Treatment with monensin alone did not improve pregnancy rate, nor did treatment with monensin enhance the pregnancy rate when a hormonal synchronisation treatment was given. Nevertheless, the use of a hormonal treatment increased pregnancy rate, suggesting that it could be used as a suitable tool to enhance the productivity in cows with marginal body condition score EEA Santiago del Estero Fil: Reineri, Pablo Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Piccardi, Mónica Belén. Instituto de Bionanotecnología del NOA (CONICET-UNSE). Laboratorio de Producción Animal; Argentina Fil: Arroquy, Jose Ignacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Fumagalli, Arnaldo Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Sumampa Coria, María. Instituto de Bionanotecnología del NOA (CONICET-UNSE). Laboratorio de Producción Animal; Argentina Fil: Hernandez, Olegario. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Santiago del Estero; Argentina Fil: Bo, Gabriel Amilcar. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina. Institute of Animal Reproduction Córdoba; Argentina Fil: Palma, Gustavo Adolfo. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina. Instituto de Bionanotecnología del NOA (CONICET-UNSE). Laboratorio de Producción Animal; Argentina

Tenderness is considered the most important meat quality trait regarding its eating quality. Post mortem meat tenderization is primarily the result of calpain mediated degradation of key proteins within muscles fibers. The calpain system originally comprised three molecules: two Ca2+-dependent proteases and a specific inhibitor. Numerous studies have shown that the calpain system plays a central role in postmortem proteolysis and meat tenderization. The objective of this review is to describe the last biochemical and molecular findings in connection with this proteolytic system and their relation with meat tenderness in bovine. Findings of DNA polymorphisms and mRNA and protein expression are described as tools to predict meat tenderness. Understanding the molecular basis of meat tenderization may be useful particularly to the meat industry and may allow amendment of pre-slaughter handling practices and postmortem treatments that improves meat quality., a terneza de la carne es considerada como el atributo de mayor importancia en el concepto de calidad de carne. El proceso de tenderización de la carne post mortem es principalmente el resultado de la degradación de proteínas clave de las fibras musculares, mediado por las proteasas del sistema calpaína. Este sistema proteico está compuesto por tres moléculas: dos proteasas calcio-dependientes y su inhibidor específico. Numerosos estudios han demostrado que el sistema calpaína desempeña un papel central en la proteólisis postmortem y en la tenderización de la carne. El objetivo de esta revisión es describir los últimos descubrimientos bioquímicos y moleculares en relación con este sistema proteolítico y su relación con la terneza de la carne bovina. Se describen los hallazgos de polimorfismos de ADN y de expresión de ARNm y proteínas, como herramientas para predecir la terneza de la carne. La comprensión de las bases moleculares de la tenderización de la carne puede ser de utilidad para la industria cárnica, permitiendo la modificación de las prácticas de manipulación antes del sacrificio y los tratamientos post mortem, mejorando la calidad de la carne bovina.

Frozen sperm is widely used in artificial insemination of cattle as well as other animal species. As a consequence of the freezing and thawing processes of semen, an excess of reactive oxygen species (ROS) are formed. ROS produce oxidative damage in sperm cells affecting both motility and fertility. Malondialdehyde (MDA) is one of the most recognized biomarkers of an advanced oxidative status. MDA was analyzed after its condensation reaction with thiobarbituric acid (TBA); however, other molecules can also react with TBA. In order to determine specifically the MDA–TBA2 condensation product in cryopreserved bovine semen, a sensitive and selective separation strategy was developed using high performance liquid chromatography (HPLC) with diode array detection (DAD). This is the first report on MDA determination in bovine semen by a separation method. Different methodological approaches were assayed. Treatment A directly measured total MDA through acidic hydrolysis combined with TBA condensation in a single step. Treatment B evaluated separately the TBA condensation product of free MDA and protein bound MDA after its release with alkaline hydrolysis. The highest concentration of MDA was detected following treatment A. An HPLC method was developed and validated by comparing with the traditional spectrophotometric method. The detection and quantification limits were 0.034 μM and 0.086 μM. The DAD response was linear in the range between 0.086 and 9.1 μM. The recovery was 91%. The intra- and inter-day relative standard deviations were 3.7 and 3.8%, respectively. The proposed HPLC method was markedly more sensitive and more specific than the traditional spectrophotometric one. Fil: Yonny, Melisa Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Reineri, Pablo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Palma, Gustavo Adolfo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Tucumán-Santiago del Estero. Estación Experimental Agropecuaria Santiago del Estero; Argentina Fil: Nazareno, Mónica Azucena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 ± 12.0%; S2, 41 ± 11.9%; S3, 39.0 ± 9.9%; S4, 42 ± 4.6%; P < 0.01; S5, 72 ± 7.1% and S6, 64 ± 9.3; P < 0.05 versus C 82 ± 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r2: >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 × 106 sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme. Fil: Palma, Gustavo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Embryo Tech S.A.; Uruguay. Reprobiotec; Argentina Fil: Olivier, N. S.. Embryo Tech S.A.; Uruguay Fil: Neumüller, Ch.. Reprobiotec; Argentina Fil: Sinowatz, F.. University of Munich; Alemania