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
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 Publikoa
Dietary 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.
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 Alimentación
Adipogenesis, 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.
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 Ekoizpena
Anatomical 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.
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.