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 - ISFOOD
Milk 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.
Open Access
Preservation of milk in liquid nitrogen during sample collection does not affect the RNA quality for RNA-seq analysis
(BMC, 2025-05-24) Jiménez Montenegro, Lucía; Alfonso Ruiz, Leopoldo; Soret Lafraya, Beatriz; Mendizábal Aizpuru, José Antonio; Urrutia Vera, Olaia; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua
Background. Standard procedures for milk sample collection for transcriptome analysis use ice as preservation method, which can afect the RNA stability and requires immediate sample processing. These problems would be eased if the milk samples could be snap-frozen in liquid nitrogen. This study describes the applicability of a new method for milk sample collection and subsequent RNA extraction from milk fat globules, determining whether the quality, integrity and quantity of the RNA extracts met the minimum requirements for downstream RNA-seq. Results. The quality of the extracts measured by A260/280 ratio and the Integrity and Quality (IQ) values obtained fulflled the reference values of 1.9 - 2.1 (P10.05) and failed to meet the RIN≥7 benchmark for RNA-seq (P>0.05). Milk fat globules contain low molecular-weight RNA fragments and minimal 18S and 28S rRNA, suggesting low RIN values were inherent to sample type. Likewise, the RNA concentration from milk fat globules were generally low (120.43±22.27 ng/µL, 102.87±15.64 ng/µL and 109.43±22.69 ng/µL, measured by Nanodrop, Qubit HS and QuanTI Ribogreen, respectively). Nevertheless, RNA-seq yielded 52.7 million paired-end reads per sample. The raw reads passed all quality control parameters having the same sequence-read lengths (151 bp), 100% base-coverage, 49% GC base content, and base quality scores of 36, enabling successful transcriptome profling. Moreover, milk proteins were identifed as the most abundant transcripts in MFG in the analysis of the most expressed genes, indicating that the sequenced reads would accurately refect the transcriptome of this milk fraction. Conclusions. Milk preservation in liquid nitrogen is a suitable sample collection method that overcomes the limitations of immediate sample processing required if ice is used. Thus, this procedure, together with the subsequent RNA isolation from milk fat globules and its sequencing by RNA-seq, would provide a practical and a non-invasive method for measuring the mammary epithelial cell transcriptome, improving the feasibility of conducting studies related to mammary gland and lactation physiology.
Open Access
An expression of mixed animal model equations to account for different means and variances in the base population
(Editions Scientifiques Medicales Elsevier, 1999) Alfonso Ruiz, Leopoldo; Estany Illa, Joan; Producción Agraria; Nekazaritza Ekoizpena
This paper presents a general expression to predict breeding values using animal models when the base population is selected, i.e. the means and variances of breeding values in the base generation differ among individuals. Rules for forming the mixed model equations are also presented. A numerical example illustrates the procedure.
Open Access
Molecular traceability of beef from synthetic Mexican bovine breeds
(FUNPEC-RP, 2011) Rodríguez Ramírez, R.; Arana Navarro, Ana; Alfonso Ruiz, Leopoldo; González Córdova, A. F.; Torrescano, G.; Guerrero Legarreta, I.; Vallejo Córdoba, B.; Producción Agraria; Nekazaritza Ekoizpena
Traceability ensures a link between carcass, quarters or cuts of beef and the individual animal or the group of animals from which they are derived. Meat traceability is an essential tool for successful identification and recall of contaminated products from the market during a food crisis. Meat traceability is also extremely important for protection and value enhancement of good-quality brands. Molecular meat traceability would allow verification of conventional methods used for beef tracing in synthetic Mexican bovine breeds. We evaluated a set of 11 microsatellites for their ability to identify animals belonging to these synthetic breeds, Brangus and Charolais/Brahman (78 animals). Seven microsatellite markers allowed sample discrimination with a match probability, defined as the probability of finding two individuals sharing by chance the same genotypic profile, of 10-8. The practical application of the marker set was evaluated by testing eight samples from carcasses and pieces of meat at the slaughterhouse and at the point of sale. The DNA profiles of the two samples obtained at these two different points in the productioncommercialization chain always proved that they came from the same animal.
Open Access
The effects of selective breeding against scrapie susceptibility on the genetic variability of the latxa black-faced sheep breed
(EDP Sciences, 2006) Alfonso Ruiz, Leopoldo; Parada Rey, Analia; Legarra, Andrés; Ugarte, Eva; Arana Navarro, Ana; Producción Agraria; Nekazaritza Ekoizpena
Breeding sheep populations for scrapie resistance could result in a loss of genetic variability. In this study, the effect on genetic variability of selection for increasing the ARR allele frequency was estimated in the Latxa breed. Two sources of information were used, pedigree and genetic polymorphisms (fifteen microsatellites). The results based on the genealogical information were conditioned by a low pedigree completeness level that revealed the interest of also using the information provided by the molecular markers. The overall results suggest that no great negative effect on genetic variability can be expected in the short time in the population analysed by selection of only ARR/ARR males. The estimated average relationship of ARR/ARR males with reproductive females was similar to that of all available males whatever its genotype: 0.010 vs. 0.012 for a genealogical relationship and 0.257 vs. 0.296 for molecular coancestry, respectively. However, selection of only ARR/ARR males implied important losses in founder animals (87 percent) and low frequency alleles (30 percent) in the ram population. The evaluation of mild selection strategies against scrapie susceptibility based on the use of some ARR heterozygous males was difficult because the genetic relationships estimated among animals differed when pedigree or molecular information was used, and the use of more molecular markers should be evaluated.
Open Access
Textile characteristics of fiber from Huacaya alpacas (Vicugna pacos)
(Universidad Nacional de Trujillo (Perú). Facultad de Ciencias Agropecuarias, 2019) Paucar Chanca, Rufino; Alfonso Ruiz, Leopoldo; Soret Lafraya, Beatriz; Mendoza Ordóñez, G.; Alvarado Quezada, F.; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Fiber from alpacas represents a substantial component of economic output for South American countries. In this study it determined the textile characteristics of fibers obtained from Huacaya alpacas raised at the South American Camelids Research and Development Center-Lachocc (CRDC-Lachocc) located at The National University of Huancavelica (UNH). Fleece samples were obtained from the mid-side rib area of 74 white alpacas (42 females and 32 males) of varying ages. The Average Fiber Diameter (AFD), Standard Deviation of the Average Fiber Diameter (SDAFD), Fiber Diameter Coefficient of Variation (FDCV), Comfort Factor (CF) and Staple Length (SL) were measured as textile characteristics and related to sex and age group. Most of the fleece samples could be classified as baby alpaca fleece according to the Peruvian Technical Standard classification (231.301.2014). Sex had no influence on any textile characteristic (p > 0.05). Meanwhile, age affected only AFD and CF (p < 0.05). Together the results indicated that alpacas farmed at CRDC-Lachocc had good potential to produce high quality fibers.
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 Ekoizpena
La 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.
Open Access
Ayuda a las decisiones de apareamiento en poblaciones animales con información genealógica incompleta
(Asociación Interprofesional para el Desarrollo Agrario, 2018) Alfonso Ruiz, Leopoldo; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD
Los coeficientes de parentesco y de relación media de parentesco son dos herramientas útiles para determinar apareamientos con el objetivo de controlar, de forma práctica, el aumento de la consanguinidad en poblaciones animales. No obstante, su utilidad se ve condicionada por la cantidad de información genealógica disponible, pues ésta determina, en ausencia de información genómica, la precisión con que podemos estimarlos. Cuando la información genealógica sea incompleta, afectando de forma distinta a los animales, la precisión de las estimaciones de los coeficientes variará entre animales, comprometiendo el resultado de las decisiones de apareamiento. El objetivo del trabajo es calcular algunas medidas de la fiabilidad de la estimación de los coeficientes de parentesco y de relación media de parentesco. Se plantea la utilización de dos medidas basadas en la proporción de antecesores conocidos en las cinco generaciones anteriores de un animal y en la incorporación de información conocida, pero no contenida en la genealogía, a través del concepto de grupos genéticos. Los resultados muestran que estas medidas pueden ayudar a mejorar las decisiones de elección de reproductores y de acoplamiento.
Open Access
DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein
(Elsevier, 2022) Alfonso Ruiz, Leopoldo; Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Bovine 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.
Open Access
Mipob: un programa de simulación para el aprendizaje en mejora genética animal
(Universidad de Córdoba, 2014) Alfonso Ruiz, Leopoldo; Producción Agraria; Nekazaritza Ekoizpena
Se ha desarrollado un programa de simulación de apoyo a la docencia de los cursos introductorios a la Mejora genética animal. El programa surgió ante la ausencia de este tipo de herramientas docentes orientadas a estudiantes de primeros cursos de grado universitario, especialmente en lengua española. El programa simula una población animal cerrada de censo reducido en la que los estudiantes deben de ir tomando las decisiones de elección de futuros reproductores y cómo aparearlos. De esta forma se facilita la introducción de los conceptos básicos sobre selección y evaluación genética, y conservación y genética de poblaciones. El programa considera el caso particular del vacuno de carne y se pueden realizar hasta un total de diez generaciones de selección. Tras haber sido probado durante dos cursos consecutivos en enseñanza reglada universitaria, está disponible de forma gratuita para su libre utilización y distribución.