Person: Alfonso Ruiz, Leopoldo
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Alfonso Ruiz
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Leopoldo
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Agronomía, Biotecnología y Alimentación
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IS-FOOD. Research Institute on Innovation & Sustainable Development in Food Chain
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0000-0002-5662-9997
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1974
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Publication Open Access Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection(Springer Nature, 2022) Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo; Azparren Domínguez, Leire; Urrutia Vera, Olaia; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute on Innovation and Sustainable Development in Food Chain - ISFOODMilk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.Publication Open Access Comparative description of growth, fat deposition, carcass and meat quality characteristics of Basque and Large White pigs(EDP Sciences, 2005) Alfonso Ruiz, Leopoldo; Mourot, Jacques; Insausti Barrenetxea, Kizkitza; Mendizábal Aizpuru, José Antonio; Arana Navarro, Ana; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako GobernuaCharacteristics of growth, fat deposition, carcass and meat quality of pigs from the Basque Black Pied breed were described and compared with those of Large White pigs. Four pens, two per breed, of eleven pigs born during the same two week period, were simultaneously fattened and slaughtered, under the same conditions. The experiment was carried out over a fixed duration (124 days) and slaughter was carried out at a fixed average age (202 days). Basque pigs showed lower growth and feed efficiency and higher backfat depth (2.6 vs. 1.7 cm, P < 0.001) than Large White pigs. The difference was especially noticeable in the middle subcutaneous fat layer (0.5 cm, P < 0.001). The meat of Basque pigs was darker, redder, more marbled, and with higher pH values than in Large White pigs. Differences in fatty acid composition were observed between breeds but they were not statistically significant (P > 0.05) because of high variability observed between animals. The Basque breed exhibited an early and higher adipose development and a higher activity of enzymes responsible for lipid synthesis than the Large White. The diameter of intramuscular adipose cells was larger in Basque (40.2 vs. 33.0 μm, P < 0.001) than in Large White pigs. The results show the particular characteristics of the Basque breed as compared to pig lines highly selected for lean growth efficiency.Publication Open Access Adipose tissue modification through feeding strategies and their implication on adipogenesis and adipose tissue metabolism in ruminants(MDPI, 2020) Urrutia Vera, Olaia; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo; Soret Lafraya, Beatriz; Insausti Barrenetxea, Kizkitza; Arana Navarro, Ana; Agronomia, Bioteknologia eta Elikadura; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD; Agronomía, Biotecnología y Alimentación; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaDietary recommendations by health authorities have been advising of the importance of diminishing saturated fatty acids (SFA) consumption and replacing them by polyunsaturated fatty acids (PUFA), particularly omega-3. Therefore, there have been efforts to enhance food fatty acid profiles, helping them to meet human nutritional recommendations. Ruminant meat is the major dietary conjugated linoleic acid (CLA) source, but it also contains SFA at relatively high proportions, deriving from ruminal biohydrogenation of PUFA. Additionally, lipid metabolism in ruminants may differ from other species. Recent research has aimed to modify the fatty acid profile of meat, and other animal products. This review summarizes dietary strategies based on the n-3 PUFA supplementation of ruminant diets and their effects on meat fatty acid composition. Additionally, the role of n-3 PUFA in adipose tissue (AT) development and in the expression of key genes involved in adipogenesis and lipid metabolism is discussed. It has been demonstrated that linseed supplementation leads to an increase in alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA), but not in docosahexaenoic acid (DHA), whilst fish oil and algae increase DHA content. Dietary PUFA can alter AT adiposity and modulate lipid metabolism genes expression, although further research is required to clarify the underlying mechanism.Publication Open Access Worldwide research trends on milk containing only A2 β-casein: a bibliometric study(MDPI, 2022) Jiménez Montenegro, Lucía; Alfonso Ruiz, Leopoldo; Mendizábal Aizpuru, José Antonio; Urrutia Vera, Olaia; Institute on Innovation and Sustainable Development in Food Chain - ISFOODThe protein fraction of β-casein may play a key role in the manifestation of a new intolerance: milk protein intolerance. The most common forms of β-casein among dairy cattle breeds are A1 and A2 β-casein. During gastrointestinal digestion of A1 β-casein, an opioid called peptide β-casomorphin-7 (BCM-7) is more frequently released, which can lead to adverse health outcomes. For that reason, novel products labelled as "A2 milk"or "A1-free dairy products" have appeared on the market. In this context, a bibliometric analysis on A2 β-casein research was carried out through the Web of Science (WoS) database. The main objective of this work was to provide an overview of the state of the art in the field of β-casein A2 by analyzing the number of publications per year, trends in thematic content, the most frequently used terms, and the most important institutions and countries in the field. This bibliometric study showed that a greater effort is needed to determine the possible implications of this novel product for human health and the market.Publication Open Access Conversión de las explotaciones de vacuno de leche a la producción de leche A2 ante una posible demanda del mercado: posibilidades e implicaciones(Asociación Interprofesional para el Desarrollo Agrario, 2019) Alfonso Ruiz, Leopoldo; Urrutia Vera, Olaia; Mendizábal Aizpuru, José Antonio; Institute on Innovation and Sustainable Development in Food Chain - ISFOODEl mercado de la leche de vaca, tras la desaparición de las cuotas lácteas, está provocando una importante restructuración en el sector. Una de las alternativas que están desarrollando, fundamentalmente las pequeñas explotaciones familiares, es la diferenciación de sus productos. En ese contexto, ha aparecido un tipo diferenciado de leche conocida como leche A2. Esta se caracteriza por estar libre de la variante A1 de la proteína β-caseína que, debido a una mayor producción de β-casomorfina-7 tras la digestión, puede dar lugar a intolerancias y afecciones gastrointestinales. En este trabajo se analizan las posibilidades e implicaciones de una selección a favor de la β-caseína A2 en el caso concreto de las explotaciones familiares de Guipúzcoa. Los resultados obtenidos a partir de 1868 hembras genotipadas muestran que la frecuencia del alelo que codifica la proteína A2 es 0,55, y no se han encontrado asociaciones desfavorables entre el alelo A2 y el valor genético para caracteres productivos, funcionales y morfológicos, ni con otros genes ni haplotipos. Bajo esta situación de partida, y empleando exclusivamente toros A2A2, la utilización o no de semen sexado, junto con las diferentes ratios de reposición que se apliquen, determinarán el periodo y el coste económico de la conversión de los rebaños a la producción de leche A2. En un escenario de costes razonables esta se estima entre los 8 y los 15 años, pudiendo llegar a suponer a una explotación de 150 vacas unos costes de unos 6.000 y 1.000 €/año, respectivamente.Publication Open Access Adiposity and adipogenic gene expression in four different muscles in beef cattle(Public Library of Science, 2017) Martínez del Pino, Lara; Arana Navarro, Ana; Alfonso Ruiz, Leopoldo; Mendizábal Aizpuru, José Antonio; Soret Lafraya, Beatriz; Producción Agraria; Nekazaritza EkoizpenaAnatomical site and divergent functionalities of muscles can be related to differences in IMF content, metabolism and adipogenic gene expression. Then, potential differences in different muscles in beef cattle were studied. As a second objective, the main sources of experimental variability associated to RT-qPCR results were analyzed following a nested design in order to implement appropriate experimental designs minimizing gene expression variability. To perform the study Longissimus thoracis (LT), Semitendinosus (SM), Masseter (MS), Sternomandibularis (ST) and subcutaneous adipose tissue (SAT) samples of Pirenaica young bulls (n = 4) were collected for IMF, collagen and protein quantification, analysis of adipocyte size distribution and gene expression (PPARG, CEBPA, FAPB4 and WNT10B). A greater IMF content was observed in MS and SM muscles, which had a bimodal adipocyte size distribution while it was unimodal in the muscles LT and ST. This suggest that the different IMF accretion in the muscles studied might be related to different rates of hyperplasia and hypertrophy and that IMF might develop later in LT and ST muscles. The former differences were not mirrored by the expression of the genes analyzed, which might be related to the different contribution of mature and non-mature adipocytes to the total gene expression. When comparing IMF and SAT gene expression, late and early developing tissues respectively, expression of PPARG, CEBPA and FABP4 was higher in the SAT, in agreement with bigger cell size and numbers. The variability study indicates that the analytical factors that add higher variability to the gene expression are the sampling and RT and therefore, it would be appropriate to include those replicates in the design of future experiments. Based on the results, the use of MS and SM muscles could allow less expensive experimental designs and bigger sample size that could permit the detection of lower relevant differences in gene expression.Publication Open Access Análisis de la relación entre el espesor de la grasa dorsal y el tamaño de los adipocitos en cerdas reproductoras(Asociación Interprofesional para el Desarrollo Agrario, 2007) Mendizábal Aizpuru, José Antonio; Abadía Durán, Silvia; Abaurrea Aramburu, Eneko; Alfonso Ruiz, Leopoldo; Producción Agraria; Nekazaritza EkoizpenaLa correcta gestión de las reservas grasas durante el ciclo productivo de las cerdas reproductoras es un factor clave para la obtención de unos buenos resultados productivos. El principal depósito graso en las cerdas lo constituye el tejido graso subcutáneo, por lo que la medición del espesor graso dorsal es un buen indicador del estado de las reservas corporales. Aunque existen diversos puntos para la medición del espesor, se recomienda hacerlo a nivel de la última costilla flotante, punto que se conoce como P2 (Quiniou, 2004). En esta localización se pueden distinguir hasta tres capas distintas de grasa subcutánea (Moody y Zobrisky, 1966). Distintos trabajos han observado un desarrollo diferencial de las tres capas durante el crecimiento de los animales (Alfonso, 2004; Fortin, 1986), así como diferencias en su actividad lipogénica (Cámara et al., 1996). Por tanto, el estudio de las características de cada capa considerada como distinto depósito graso puede ser importante para conocer los mecanismos de acumulación y movilización de las reservas grasas. En este sentido, en el presente trabajo se estudia la relación entre el espesor de la grasa y el tamaño de los adipocitos de cada una de las capas descritas en la grasa subcutánea dorsal.Publication Open Access DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein(Elsevier, 2022) Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaBovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in the dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify the A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. The results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10−5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for the A1 allele in A2 samples at 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify the A1 allele absence in “A2 milk” by screening commercial products on the market.Publication Open Access Expression of key myogenic, fibrogenic and adipogenic genes in Longissimus thoracis and masseter muscles in cattle(Cambridge University Press, 2020) Martínez del Pino, Lara; Urrutia Vera, Olaia; Arana Navarro, Ana; Alfonso Ruiz, Leopoldo; Mendizábal Aizpuru, José Antonio; Soret Lafraya, Beatriz; Agronomia, Bioteknologia eta Elikadura; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD; Agronomía, Biotecnología y AlimentaciónAdipogenesis, myogenesis and fibrogenesis are related processes that can contribute to meat quality. Therefore, extending the knowledge of these processes would facilitate the identification of molecular markers that predict intramuscular fat accretion. The main purpose of this work, based on previous results, was to further study the expression of key genes related to adipogenic, myogenic, fibrogenic processes and some cytokines in Longissimus thoracis (LT) and Masseter (MS) muscles of Pirenaica and Holstein young bulls. Longissimus thoracis and MS muscles from Pirenaica (n = 4) and Spanish Holstein (n = 4) were sampled for proximate analysis, determination of adipocyte size distribution and expression of key candidate genes. Fat percentage was lower in LT than in MS muscle in Pirenaica young bulls (P = 0.023) and was higher in LT muscle in Holstein than in Pirenaica young bulls (P = 0.007). Gene expression analysis revealed that the mRNA level of myogenic differentiation 1 (MYOD) was higher in LT than in MS muscles in both groups of animals (P < 0.001) and that myostatin (MSTN) expression was also higher in LT than in MS muscle in Holstein bulls (P = 0.001). On the other hand, MSTN and PPARG showed higher expression in LT and MS in Pirenaica young bulls (P = 0.026), while the expression of fatty acid-binding protein 4 (FABP4) was higher in Holstein young bulls, also in both muscles (P < 0.001). The results suggested that the development of intramuscular adipose depot was directly related to the expression of adipogenic genes, such as FABP4, but inversely related to the expression of the cytokine MSTN and the myogenic gene MYOD, genes which showed a muscle-specific expression.Publication Open Access Caracterización de la distribución del tamaño de los adipocitos para el estudio del tejido adiposo en producción animal(Asociación Interprofesional para el Desarrollo Agrario, 2016) Alfonso Ruiz, Leopoldo; Mendizábal Aizpuru, José Antonio; Producción Agraria; Nekazaritza EkoizpenaEl tamaño de los adipocitos, principales células constituyentes del tejido adiposo, ha sido objeto de numerosos estudios por el hecho de estar relacionado con el grado de desarrollo y la actividad metabólica de ese tejido. Su análisis resulta en ocasiones complejo dada la bimodalidad de su distribución. Este trabajo presenta un método de análisis basado en contrastar, en primer lugar la unimodalidad de la distribución del tamaño de los adipocitos, frente a una distribución bimodal. Posteriormente propone algunos parámetros para describir adecuadamente la distribución bimodal. Para su aplicación se desarrolló un sencillo programa informático que se utiliza, a modo de ejemplo, para analizar los datos de dos trabajos, previamente publicados, sobre el desarrollo adipocitario. Los resultados muestran el interés de utilizar contrastes de bimodalidad frente a la mera inspección visual de los histogramas de distribución. Además, cuando la hipótesis de unimodalidad es rechazada, la utilización de parámetros descriptivos de bimodalidad, como el porcentaje de adipocitos por encima del punto de inflexión entre ambas modas, permite una comparación más adecuada entre tratamientos experimentales.