Effect of animal mixing as a stressor on biomarkers of autophagy and oxidative stress during pig muscle maturation

The objective of this work was to study the postmortem evolution of potential biomarkers of autophagy (Beclin 1, LC3-II/LC3-I ratio) and oxidative stress (total antioxidant activity, TAA; superoxide dismutase activity, SOD and catalase activity, CAT) in the Longissimus dorsi muscle of entire male ((Large White × Landrace) × Duroc) pigs subjected to different management treatments that may promote stress, such as mixing unfamiliar animals at the farm and/or during transport and lairage before slaughter. During the rearing period at the farm, five animals were never mixed after the initial formation of the experimental groups (unmixed group at the farm, UF), whereas 10 animals were subjected to a common routine of being mixed with unfamiliar animals (mixed group at the farm, MF). Furthermore, two different treatments were used during the transport and lairage before slaughter: 10 pigs were not mixed (unmixed group during transport and lairage, UTL), whereas five pigs were mixed with unfamiliar animals on the lorry and during lairage (mixed group during transport and lairage, MTL). These mixing treatments were then combined into three pre-slaughter treatments – namely, UF-UTL, MF-UTL and MF-MTL. The results show that MF-UTL and MF-MTL increased significantly the muscle antioxidant defense (TAA, SOD and CAT) at short postmortem times (4 and 8 h; P < 0.001), followed by an earlier depletion of the antioxidant activity at 24 h postmortem (P < 0.05). We also found that mixing unfamiliar animals, both at the farm and during transport and lairage, triggers postmortem muscle autophagy, which showed an earlier activation (higher expression of Beclin 1 and LC3-II/LC3-I ratio at 4 h postmortem followed by a decreasing pattern of this ratio along first 24 h postmortem) in the muscle tissues of animals from the MF-UTL and MF-MTL groups, as an adaptive strategy of the muscle cells for counteracting induced stress. From these results, we propose that monitoring the evolution of the main biomarkers of autophagy (Beclin 1, LC3-II/LC3-I ratio) and muscle antioxidant defense (TAA, SOD, CAT) in the muscle tissue within the first 24 h postmortem may help the detection of animal stress and its potential effect on the postmortem muscle metabolism.info:eu-repo/semantics/publishedVersio

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Enzimas antioxidantes en la maduración de carne de vacuno procedente de dos cabañas autóctonas asturianas

Antioxidant enzymes throughout ageing from two local cattle breeds In this study, meat from double-muscled (“culones”, mh/mh) and normal (+/+) young bulls of the breed Asturiana de los Valles (AV) as well as normal (+/+) animals from Asturiana de la Montaña (AM) was analysed during ageing at 4°C with regard to its oxidative status. Activities of the following main antioxidant enzymes were determined: Superoxide dismutase (SOD), Catalase (CAT) and Glutathione reductase (GR). In both breeds (AV and AM), lipid peroxidation (LPO) was also measured throughout the ageing period, because meat shelf life is mainly limited by lipids and proteins. All enzymes were apparently stable during meat ageing. While GR exhibited the lowest activity among antioxidant enzymes, SOD and CAT showed activity along the ageing period studied, with similar activity pattern seen in both double-muscled and normal animals, although, being advanced in the formers. In normal animals of both the AV and AM breeds, lipid peroxidation did not vary during ageing. However, double- muscled meat showed a significant increase after 7 days of maturation.En este trabajo se determinó el estado oxidativo de la carne procedente de terneros añojos de genotipo culón (mh/mh) y normal (+/+) de la raza Asturiana de los Valles (AV) así como terneros normales (+/+) de la raza Asturiana de la Montaña (AM) durante su periodo de maduración a 4ºC, mediante el estudio de la actividad de los principales enzimas antioxidantes: Superóxido dismutasa (SOD), Catalasa (CAT) y Glutatión reductasa (GR). Se midió también el nivel de peroxidación lipídica (LPO) a lo largo de la maduración a 4ºC de carne procedente de ambas razas (AV y AM) por ser los lípidos y las proteínas las biomoléculas que más limitan la vida media de la carne durante este periodo. Los resultados reflejaron una clara estabilidad de los enzimas durante el periodo de maduración de la carne, siendo el enzima GR el que mostró menor actividad, mientras que SOD y CAT presentaron actividad a lo largo de todo el período de maduración estudiado con un patrón de actividad que se repitió en la carne de terneros (mh/mh) y (+/+) de raza AV, siendo más adelantado en los primeros que en los segundos. La peroxidación lipídica no mostró variación alguna a lo largo de la maduración tanto en animales (+/+) de la raza AV como en animales (+/+) de la raza AM. Sin embargo, los animales (mh/mh) de la raza AV mostraron un incremento significativo del daño lipídico en el músculo tras 7 días de maduración