Claudia Terlouw

Skeletal muscle proteomics in livestock production

Proteomics allows studying large numbers of proteins, including their post-translational modifications. Proteomics has been, and still are, used in numerous studies on skeletal muscle. In this article, we focus on its use in the study of livestock muscle development and meat quality. Changes in protein profiles during myogenesis are described in cattle, pigs and fowl using comparative analyses across different ontogenetic stages. This approach allows a better understanding of the key stages of myogenesis and helps identifying processes that are similar or divergent between species. Genetic variability of muscle properties analysed by the study of hypertrophied cattle and sheep are discussed. Biological markers of meat quality, particularly tenderness in cattle, pigs and fowl are presented, including protein modifications during meat ageing in cattle, protein markers of PSE meat in turkeys and of post-mortem muscle metabolism in pigs. Finally, we discuss the interest of proteomics as a tool to understand better biochemical mechanisms underlying the effects of stress during the pre-slaughter period on meat quality traits. In conclusion, the study of proteomics in skeletal muscles allows generating large amounts of scientific knowledge that helps to improve our understanding of myogenesis and muscle growth and to control better meat quality.

Presence of cues from stressed conspecifics increases reactivity to aversive events in cattle : Evidence for the existence of alarm substances in urine

In gregarious species like cattle, the presence of partners often affects fear-related reactions. The first experiment investigated whether behavioral and physiological responses to stress depend on the emotional state of the partner. Aubrac heifers were presented with food in a novel environment. Compared to heifers in the presence of a companion that had been previously habituated to the environment without receiving shocks, those in the presence of a companion animal that had previously received electric shocks in that environment had a stronger increase of cortisol response (11.2+/-1.1 vs. 7.4+/-0.9 ng/mL), showed a significantly longer latency to feed (60.1+/-12.3 vs. 17.8+/-5.9 s), and fed more slowly (54.5+/-11.4 vs. 110+/-7.3 s). After repeated exposure to the test conditions, when heifers of both treatments fed rapidly after entrance, response to an unexpected air blast from the feeding bucket was measured. Heifers in the presence of a stressed companion showed an increased latency to feed again compared to those with a nonstressed companion (44.5+/-5.1 vs. 22.8+/-4.3 s). Stressed companions urinated during tests; therefore the second experiment investigated whether heifers respond differently to urine collected from stressed and nonstressed conspecifics. In a first test, heifers were presented with food on a grid in a bucket in a novel environment. They had a longer latency to feed when the bucket contained urine from stressed rather than from nonstressed conspecifics underneath the grid (128.1+/-9.6 vs. 108.2+/-4.9 s). In a second test, heifers were presented with a novel object in a familiar environment. Heifers showed a longer latency to explore the object when it had been sprayed with urine from stressed compared to urine from nonstressed conspecifics (215.2+/-45.0 vs. 25.8+/-8.6 s). The results show that heifers perceive the state of increased stress of conspecifics and become more fearful as a result. They further show that this perception is at least partly mediated by olfactory cues in the urine.

Pig Longissimus lumborum proteome: Part II: Relationships between protein content and meat quality

Gender, rearing environment and breed of sire influenced 50.5% of the matched protein spots of the soluble fraction and some meat quality traits [Kwasiborski, A., Sayd, T., Chambon, C., Santé-Lhoutellier, V., Rocha, D., & Terlouw, C. (2008). Muscle proteome in pigs: Part I: Effects of genetic background, rearing environment and gender. Meat Science]. Multiple regression analyses determined that 1 or 2 proteins explained between 24% and 85% of variability in Longissimus meat quality. Regression models differed between treatment groups, but relationships between proteins and meat quality traits seemed to be related to common underlying mechanisms. Thus, proteins retained in models for ultimate pH, lightness, drip, thawing and cooking loss were related to the glycolytic pathway, phosphate transfer, or fibre type composition. Another model for thawing loss retained proteins related to denaturation of myofibrils or lipid content. The models for redness involved proteins related to post-mortem oxidative activity. Thus, proteins correlated with meat quality traits were related to biochemical mechanisms known to be involved in meat quality. Relative contributions of these mechanisms may vary according to gender, sire breed or rearing environment.

Differences in Pig Muscle Proteome According to HAL Genotype: Implications for Meat Quality Defects

Bidimensional electrophoresis was used to compare sarcoplasmic protein profiles of early post-mortem pig semimembranosus muscles, sampled from pigs of different HAL genotypes (RYR1 mutation 1841T/C): 6 NN, 6 Nn, 6 nn. ANOVA showed that 55 (18%) of the total of 300 matched protein spots were influenced by genotype, and hierarchical clustering analysis identified 31 (10% of the matched proteins) additional proteins coregulated with these proteins. Fold-changes of differentially expressed proteins were between 1.3 and 21.8. Peptide mass fingerprinting identification of 78 of these 86 proteins indicates that faster pH decline of nn pigs was not explained by higher abundance of glycolytic enzymes. Results indicate further that nn muscles contained fewer proteins of the oxidative metabolic pathway, fewer antioxidants, and more protein fragments. Lower abundance of small heat shock proteins and myofibrillar proteins in nn muscles may at least partly be explained by the effect of pH on their extractability. Possible consequences of lower levels of antioxidants and repair capacities, increased protein fragmentation, and lower extractability of certain proteins in nn muscles on meat quality are discussed.

Identifying and monitoring pain in farm animals: a review

One important objective for animal welfare is to maintain animals free from pain, injury or disease. Therefore, detecting and evaluating the intensity of animal pain is crucial. As animals cannot directly communicate their feelings, it is necessary to identify sensitive and specific indicators that can be easily used. The aim of the present paper is to review relevant indicators to assess pain in several farm species. The term pain is used for mammals, birds and fish, even though the abilities of the various species to experience the emotional component of pain may be different. Numerous behavioural changes are associated with pain and many of them could be used on farms to assess the degree of pain being experienced by an animal. Pain, as a stressor, is associated with variations in the hypothalamic-pituitary-adrenal axis as well as in the sympathetic and immune systems that can be used to identify the presence of pain rapidly after it started. However, most of these measures need sophisticated equipment for their assessment. Therefore, they are mainly adapted to experimental situations. Injuries and other lesional indicators give information on the sources of pain and are convenient to use in all types of situations. Histopathological analyses can identify sources of pain in experimental studies. When pronounced and/or long lasting, the pain-induced behavioural and physiological changes can decrease production performance. Some indicators are very specific and sensitive to pain, whereas others are more generally related to stressful situations. The latter can be used to indicate that animals are suffering from something, which may be pain. Overall, this literature review shows that several indicators exist to assess pain in mammals, a few in birds and very few in fish. Even if in some cases, a single indicator, usually a behavioural indicator, may be sufficient to detect pain, combining various types of indicators increases sensitivity and specificity of pain assessment. Research is needed to build and validate new indicators and to develop systems of pain assessment adapted to each type of situation and each species.

Pig Longissimus lumborum proteome: Part I. Effects of genetic background, rearing environment and gender

A 2×2×2 factorial experiment on Longissimus lumborum of 24 pigs found that rearing environment (indoors or outdoors), breed of sire (Duroc or Large White), and gender (female or castrated male) influenced 22, 10, and 88 proteins of the soluble fraction, respectively, containing 220 matched spots in total. Some proteins were influenced by more than one main effect. Outdoor rearing resulted in lower levels of enzymes of the glycolytic pathway suggesting a more oxidative metabolism. Breed of sire slightly altered the balance of enzymes of the glycolytic pathway. Gender had profound effects. In particular, different enzyme levels suggest a more lipid oriented energy metabolism, and a higher extractability of myofibrillar proteins suggest altered control of the contractile apparatus, in castrated males. Differences in extractability did not explain the profound gender effects. Glycogen content, ultimate pH, drip and thawing losses showed main or interactive effects of the three treatment factors.

Meta-analysis of the effects of dietary vitamin E supplementation on α-tocopherol concentration and lipid oxidation in pork

Meta-analyses have been carried out to quantify the effect of dietary vitamin E on α-tocopherol accumulation and on lipid oxidation in porcine M. longissimus. Published results of 13 (vitamin E accumulation) and 10 (lipid oxidation) experiments respectively were used for the analyses. After a number of standardization procedures, a nonlinear relationship was found between the supplementary vitamin E and the accumulation of α-tocopherol in pork which approached a maximum value of 6.4 μg/g tissue. Pork lipid oxidation levels were described in terms of Thiobarbituric Acid Reacting Substances (TBARS) values. The statistical analysis revealed significant effect of vitamin E dose, muscle α-tocopherol concentration and supplementation time on TBARS, resulting in two prediction models for lipid oxidation. Meta-analysis has proven to be a valuable tool for combining results from previous studies to quantify the effects of dietary vitamin E. Further studies, carried out with standardized experimental protocols would be beneficial for model validation and to increase the predictive power of the derived models.

Alternative rearing systems in pigs: consequences on stress indicators at slaughter and meat quality

The objective of this study was to evaluate the effects of three alternative (ALT) rearing systems for growing pigs (outdoor: 150 m2/pig; straw bedding: 1.30 m2/pig; and hut with access to a courtyard: 1.30 m2/pig) compared with a conventional system (fully slatted floor: 0.65 m2/pig, considered as control), on pre-slaughter stress indicators in relation with meat quality. To that end, the number of skin lesions on whole carcasses, as well as blood creatine kinase (CK) activity and urine levels in cortisol and catecholamines (adrenaline and noradrenaline) were determined at slaughter. Glycolytic potential (GP) and ultimate pH of the semimembranosus muscle were also measured. The global correlation network calculated between all these parameters shows that the indicators of pre-slaughter muscle activity (plasma CK) and/or stress indicators (e.g. adrenaline) are negatively (r=-0.26, P<0.01; r=-0.29, P<0.05, respectively) correlated with muscle GP and positively (r=0.17, P<0.05; r=0.44, P<0.001, respectively) with meat ultimate pH. Although some traits measured were sensitive to the degree of pre-slaughter mixing, they differed across rearing systems. The differences were most pronounced for the comparison of outdoors v. slatted floor. The lower levels of plasma CK and urinary catecholamines, and the lower number of carcass skin lesions of pigs reared outdoors, were related to a lower meat ultimate pH. Thus, ALT rearing systems influence animal welfare and meat quality, by providing enriched environmental conditions to the animals.

Bayesian meta-analysis of the effect of fasting, transport and lairage times on four attributes of pork meat quality

Technological meat quality is a significant economic factor in pork production, and numerous publications have shown that it is strongly influenced both by genetic status and by rearing and slaughter conditions. The quality of meat is often described by meat pH at different times postmortem, as well as by color and drip loss. A meta-analysis based on a database built from 27 studies corresponding to a total of 6526 animals classified was carried out. The purpose of this meta-analysis was to study the effect of fasting, lairage and transport durations on four main attributes of the technological pork meat quality. A Bayesian hierarchical meta-regression approach was adopted. The results of our meta-analysis showed that fasting time had a significant effect on pH measured 24h post-mortem (pHu) and drip loss (DL) measured in longissimus muscle. While, lairage affected only the pHu in semimembranosus muscle. Interestingly, we found that DL was the lone attribute that was affected by transport time and its interaction with fasting time.

Consciousness, unconsciousness and death in the context of slaughter. Part II. Evaluation methods

This second review describes indicators of consciousness and unconsciousness that can be used in the abattoir. These indicators evaluate different aspects of cerebral functioning, but only indirectly. It is therefore necessary to monitor several indicators. Animals are considered unconscious if signs of consciousness are absent, and signs of unconsciousness are present. Given that the unconscious state may be reversible it is further necessary to monitor these indicators until the end of bleeding. The techniques used to diagnose brain death in humans cannot be used in the slaughterhouse. Under field conditions, at the end of bleeding, the absence of breathing and of brainstem reflexes and the adequacy of the exsanguination are verified. If these three aspects are confirmed, in the context of the slaughterhouse and at this stage of the slaughter process the loss of vital functions is irreversible and the animal can be considered dead.