Fear, anxiety, and production in laying hens with healed keel bone fractures.

For laying hens, the immediate aftermath and healing period of a keel fracture (KF) is characterized by reduced ability to perform species-specific behavior, access resources, and pain. However, the longer-term impacts, once the fracture is completely healed, are less clear. As well as acute pain and behavioral changes, a negative experience can shape future responses to putatively threatening stimuli, raising future fear, and anxiety levels during husbandry-related events. We aimed to determine whether hens that had previously sustained keel bone fractures, but were now outside of the peak age range for new fractures, showed higher fear and anxiety levels compared to intact hens. We also determined if healed keel bone fractures were associated with reduced production, changes in behavior and resource use. One hundred and fifty hens with a palpation score of 1 ( "KF") and 150 hens with a palpation score of 0 (keel fracture free, "KFF") were selected from a commercial farm at 63 wk of age and housed in 6 groups (3 × KF and 3 × KFF). We compared production (hen weight and feed consumption, egg quantity, quality and weight, floor eggs, shell thickness, and weight) and home pen behavior (behaviors and transitional movements) in both groups. Finally, we measured the responses of KF (n = 75) and KFF (n = 75) during tonic immobility, novel arena, and novel object tests. KF and KFF hens did not differ in their responses to the tonic immobility, novel arena, and novel object tests, nor were there differences between the 2 groups in home pen behavior and transitional movements. KFF birds were lighter and laid eggs with less eggshell membrane compared KF birds, but no differences were found between KF and KFF in any other production measures. We found no evidence that healed KFs were associated with detrimental welfare effects in laying hens, but further work is required to determine the mechanisms and implications of the lower body weight and egg shell membrane.

Light-based monitoring devices to assess range use by laying hens.

Access to an outdoor range has many potential benefits for laying hens but range use can be poor due to factors only partly understood. Techniques to monitor individual range use within commercial flocks are crucial to increase our understanding of these factors. Direct observation of individual range use is difficult and time-consuming, and automatic monitoring currently relies on equipment that is difficult to use in an on-farm setting without itself influencing range use. We evaluated the performance of a novel small, light and readily portable light-based monitoring system by validating its output against direct observations. Six commercial houses (2000 hens/house) and their adjacent ranges were used, three of which were equipped with more structures on the range than the others (to determine whether cover would influence monitoring accuracy). In each house, 14 hens were equipped with light monitoring devices for 5 discrete monitoring cycles of 7 to 8 consecutive days (at 20, 26, 32, 36 and 41 weeks of age). Light levels were determined each minute: if the reading on the hen-mounted device exceeded indoor light levels, the hen was classified as outside. Focal hens were observed directly for 5 min/hen per week. Accuracy (% of samples where monitoring and direct observations were in agreement) was high both for ranges with more and with fewer structures, although slightly better for the latter (92% v. 96% ± 1 SEM, F1,19 = 5.2, P = 0.034). Furthermore, accuracy increased over time (89%, 94%, 95%, 98% ± 1 SEM for observations at 26, 32, 36 and 41 weeks, respectively, F3,19 = 3.2, P = 0.047), probably due to progressively reduced indoor light levels resulting from partial closing of ventilation openings to sustain indoor temperature. Light-based monitoring was sufficiently accurate to indicate a tendency for a greater percentage of monitored time spent outside when more range structures were provided (more: 67%, fewer: 56%, SEM: 4, $\chi_1^2 = 2.9$, P = 0.089). Furthermore, clear and relatively consistent individual differences were detected. Individuals that were caught outside at the start of the experiment ranged more throughout its duration (caught outside: 72%, caught inside 51%, SEM: 4, $\chi_1^2 = 10.0$, P = 0.002), and individual range use was correlated between monitoring cycles (for adjacent monitoring cycles: $r_s^2 = 0.5-0.7$, P < 0.0001). This emphasizes the importance of studying range use on an individual level. In conclusion, our light-based monitoring system can assess individual range use accurately (although accuracy was affected by house characteristics to some extent) and was used to show that both cover availability and individual characteristics affected range use.

High-fat, high-fructose, high-cholesterol feeding causes severe NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine.

Pediatric obesity and nonalcoholic steatohepatitis (NASH) are on the rise in industrialized countries, yet our ability to mechanistically examine this relationship is limited by the lack of a suitable higher animal models. Here, we examined the effects of high-fat, high-fructose corn syrup, high-cholesterol Western-style diet (WD)-induced obesity on NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine. Juvenile female Ossabaw swine (5 wk old) were fed WD (43.0% fat; 17.8% high-fructose corn syrup; 2% cholesterol) or low-fat diet (CON/lean; 10.5% fat) for 16 wk ( n = 6 each) or 36 wk ( n = 4 each). WD-fed pigs developed obesity, dyslipidemia, and systemic insulin resistance compared with CON pigs. In addition, obese WD-fed pigs developed severe NASH, with hepatic steatosis, hepatocyte ballooning, inflammatory cell infiltration, and fibrosis after 16 wk, with further exacerbation of histological inflammation and fibrosis after 36 wk of WD feeding. WD feeding also resulted in robust cecal microbiota changes including increased relative abundances of families and genera in Proteobacteria ( P < 0.05) (i.e., Enterobacteriaceae, Succinivibrionaceae, and Succinivibrio) and LPS-containing Desulfovibrionaceae and Desulfovibrio and a greater ( P < 0.05) predicted microbial metabolic function for LPS biosynthesis, LPS biosynthesis proteins, and peptidoglycan synthesis compared with CON-fed pigs. Overall, juvenile Ossabaw swine fed a high-fat, high-fructose, high-cholesterol diet develop obesity and severe microbiota dysbiosis with a proinflammatory signature and a NASH phenotype directly relevant to the pediatric/adolescent and young adult population.

Cocaine self-administration and reinstatement in female rats selectively bred for high and low voluntary running.

BACKGROUND: Previous research has found that rats behaviorally screened for high (vs. low) wheel running were more vulnerable to cocaine abuse. To assess the extent to which a genetic component is involved in this drug-abuse vulnerability, rats selectively bred for high or low voluntary running (HVR or LVR, respectively) were examined for differences in cocaine seeking in the present study. METHODS: Female rats were trained to lever press for food and then were assessed for differences in acquisition of cocaine (0.4mg/kg; i.v.) self-administration across 10 sessions. Once acquired, rats self-administered cocaine for a 14-day maintenance phase, followed by a 14-day extinction phase when cocaine was no longer available. Subsequently, reinstatement of cocaine seeking was examined with priming injections of cocaine (5, 10 & 15mg/kg), caffeine (30mg/kg), yohimbine (2.5mg/kg) and cocaine-paired cues. RESULTS: A greater percentage of LVR rats met the acquisition criteria for cocaine self-administration and in fewer sessions than HVR rats. No differences in responding for cocaine were observed between phenotypes during maintenance. However, during extinction LVR rats initially responded at higher rates and persisted in cocaine seeking for a greater number of sessions. No phenotype differences were observed following drug and cue-primed reinstatement of cocaine seeking. CONCLUSIONS: In general, LVR rats were more sensitive to the reinforcing effects of cocaine than HVR rats during periods of transition into and out of cocaine self-administration. Thus, LVR rats sometimes showed a greater vulnerability cocaine seeking than HVR rats.

Muscle adaptation to exercise: New Saltin's paradigms.

The title assigned for my lecture at the Saltin Symposium was "Muscle adaptation to exercise: new paradigms." The title's topic made me remember that some of Saltin's paradigms for his development of a novel exercise model were either originated by him or modified by him from existing information. Therefore, I deemed it would be instructive for future generations to consider one facet of his 54-year career--human exercise models. I arbitrarily selected to share five examples of new paradigm models initiated by Saltin. They are: bed rest; arms vs legs; one leg vs the other leg; myokine communication from skeletal muscle to other organs/tissues; and 42-day cross-country skiing expedition. I arbitrarily selected the above as examples of novel approaches that he used to the study humans during maximal endurance exercise. Noteworthy though is that Saltin's lifetime demeanor, itself, is a model for other scientists. In final analysis, the world is richer due to his passion to study humans to advance medical science by uncovering mechanisms as to how the human body is constructed to perform endurance types of exercise at maximal intensities and durations.

The effects of long (C20/22) and short (C18) chain omega-3 fatty acids on keel bone fractures, bone biomechanics, behavior, and egg production in free-range laying hens.

Keel fractures in the laying hen are the most critical animal welfare issue facing the egg production industry, particularly with the increased use of extensive systems in response to the 2012 EU directive banning conventional battery cages. The current study is aimed at assessing the effects of 2 omega-3 (n3) enhanced diets on bone health, production endpoints, and behavior in free-range laying hens. Data was collected from 2 experiments over 2 laying cycles, each of which compared a (n3) supplemented diet with a control diet. Experiment 1 employed a diet supplemented with a 60:40 fish oil-linseed mixture (n3:n6 to 1.35) compared with a control diet (n3:n6 to 0.11), whereas the n3 diet in Experiment 2 was supplemented with a 40:60 fish oil-linseed (n3:n6 to 0.77) compared to the control diet (n3:n6 to 0.11). The n3 enhanced diet of Experiment 1 had a higher n3:n6 ratio, and a greater proportion of n3 in the long chain (C20/22) form (0.41 LC:SC) than that of Experiment 2 (0.12 LC:SC). Although dietary treatment was successful in reducing the frequency of fractures by approximately 27% in Experiment 2, data from Experiment 1 indicated the diet actually induced a greater likelihood of fracture (odds ratio: 1.2) and had substantial production detriment. Reduced keel breakage during Experiment 2 could be related to changes in bone health as n3-supplemented birds demonstrated greater load at failure of the keel, and tibiae and humeri that were more flexible. These results support previous findings that n3-supplemented diets can reduce fracture likely by increasing bone strength, and that this can be achieved without detriment to production. However, our findings suggest diets with excessive quantities of n3, or very high levels of C20/22, may experience health and production detriments. Further research is needed to optimize the quantity and type of n3 in terms of bone health and production variables and investigate the potential associated mechanisms.

Non-passaged muscle precursor cells from 32-month old rat skeletal muscle have delayed proliferation and differentiation.

OBJECTIVES: The systemic environment and satellite cell dysfunction have been proposed as important contributors in the development of sarcopenia and impaired skeletal muscle regrowth with ageing. In the present study, we investigated effects of serum age on proliferation of muscle precursor cells (MPCs) isolated from skeletal muscles of young and old rats. MATERIALS AND METHODS: We examined proliferation and subsequent differentiation of non-passaged MPCs isolated from skeletal muscles of 1-, 3- and 32-month old rats over a 72-h time course, using a serum cross-over design. RESULTS AND CONCLUSIONS: We found no effect of serum age on MPC proliferation, but we did discover that MPCs isolated from skeletal muscle of 32-month old rats had delayed onset of, and exit from proliferation, compared to MPCs isolated from skeletal muscle of 1-month old rats. Delayed proliferation of MPCs from 32-month old rats was associated with delayed p38 MAPK phosphorylation, and MyoD and p21(Cip1) protein expression. We also demonstrate that MPCs from 32-month old rats exhibited lower levels of muscle creatine kinase mRNA compared to 1-month old rats, but elevated levels of myogenin mRNA, when stimulated to differentiate after 36 h proliferation. These findings suggest that delayed entry and exit of the cell cycle observed in MPCs from 32-month old rats may compromise their ability to respond to differentiation stimuli and subsequently impair myogenic potential of 32-month old skeletal muscle, in this model.

Pop hole use by hens with different keel fracture status monitored throughout the laying period.

The aim of this work was to study the effect of keel fractures on the extent to which free-range hens access the range through pop holes. Over two consecutive laying periods (two production years) a total of 1100 individual birds from one half of a house, divided into four separated flocks, were caught at 25, 35, 45, 55 and 65 weeks, palpated to assess the prevalence and severity of keel fractures and tagged with RFID transponders. Their use of pop holes was subsequently monitored in some cases from week 25 to end of lay at 68 to 70 weeks. At regular intervals (every 10 weeks), the tagged birds were re-caught to assess changes in keel fracture prevalence and severity. The average percentage of birds with fractured keels at 25, 35, 45, 55, 65 and at end of lay (68 to 70 weeks of age) was 5.5, 25.5, 49, 63, 66.5 and 78.5, respectively, across both production years. The effect of keel score on pop hole use was modelled statistically, adjusting for weather conditions and age of the birds. There were significant effects of most of the weather variables recorded, as well as age of the bird, on use of pop holes and also a significant effect of keel score. Higher keel scores resulted in a reduction in pop hole use. A significant statistical interaction between keel score and ambient temperature revealed an accelerated reduction in use as the temperature decreased and keel score increased. It is concluded that the occurrence of keel fractures may affect the birds' ability or willingness to utilise the outdoor range provided by free-range housing systems, thereby reducing the potential welfare advantages of this type of housing.

Continuous monitoring of pop hole usage by commercially housed free-range hens throughout the production cycle.

Free-range laying hens are able to move between the indoor house and range through exits termed pop holes. The aim of this study was to examine the proportion of the flock that used the pop holes and to identify patterns of movement throughout the flock cycle. Four flocks of free-range hens each of 1500 birds were studied. Ten per cent of each flock were tagged with RFID (radio-frequency identification) transponders and their pop hole activity studied throughout the production cycle. Within two weeks of tagging at 25, 35, 45, 55 and 65 weeks of age, approximately 80 per cent of the tagged birds were seen in the pop holes and 50 per cent of the tagged birds were seen on 80 per cent of the days available to them after tagging. Within the flock, subpopulations of birds could be identified: those that never ventured to the pop holes (approximately 8 per cent), those that used the pop holes very infrequently (approximately 8 per cent), those that sat in the pop holes (approximately 4 per cent), and those that used the pop holes frequently (approximately 80 per cent). There was an effect of age of the birds, time of day and daily mean temperature on pop hole usage. Additional factors affecting activity on particular days were wind speed, rainfall and hours of sunshine. The findings show that a significant proportion of the flock accesses the pop holes on a regular basis with only a very small proportion preferring to stay in the house.

The future: genes, physical activity and health.

The assigned title for the Lindhard presentation was to examine the future of genes, physical activity and health. The current review is a summary of this presentation. Caution is expressed that technology is improving so rapidly that a future view is limited to a few years as opposed to the 100 years passing since Lindhard's achievements. The near futuristic opportunities and challenges for four major topic topics are reviewed here. Concerns are expressed over current usage of the terms 'control' group and 'non-responders' in exercise research. Our view is that 'control' needs to be differentiated between its usage for treatments of exercise to restore natural functions in individuals with less than healthy levels of physical activity and the inherited genome's expectation for physical activity levels to maintain normal function. For the second discussed topic, it is proposed that the term 'non-responders' should be replaced by the term 'low sensitivity' as there may be no such human who is a non-responder to every exercise adaptation. The third futuristic topic is exercise prescription as envisioned for individualized medicine. However, numerous limitations and challenges exist to truly optimal exercise medicine at the level of one individual. Finally, preventative physical activity medicine is discussed. Physical activity as a therapy now exists to prevent most of the chronic diseases. The future needs to understand the molecular basis for how the body becomes dysfunctional when its level of physical activity does not match the norm of physical activity that selected our inherited genome.

Basic concepts about genes, inactivity and aging.

Remarkably, 80-year-old humans who have partaken in lifelong aerobic or strength training have maximal aerobic capacities or muscle strengths comparable with that of sedentary individuals aged 50 or 55-year-old, respectively. Such delays in functional aging are clinically significant because lower aerobic and lower strength capacities increase the risk of premature death. In this short review, we speculate that the lack of daily physical activity induces evolutionarily selected mechanisms to use or lose, one of which is related to nutritional status.

Lack of adequate appreciation of physical exercise's complexities can pre-empt appropriate design and interpretation in scientific discovery.

Two major issues are presented. First, a challenge is made by us that a misunderstanding of physiology has led to incomplete or wrong functional designations of genes in some cases. Normal physiological processes are dynamic, integrated and periodic, and, therefore, it is difficult to define normal physiological function by looking at a single time point or single process in a non-stressed subject. The ability of the organism to successfully respond to homeostatic disruptions defines normal physiology. Genes were selected for survival and to appropriately respond to stresses, such as physical activity. Omitting gene functions by restricting them to non-stressful conditions could lead to less than optimal primary preventions, treatments and cures for diseases. Physical exercise, as a stressor, should be used to better demonstrate the complete functional classifications of some genes. Second, the challenge from others of an 'exercise pill' as a mimetic of natural physical activity will be shown to be lacking a scientific basis. The concept of an 'exercise pill'/'exercise mimetic' demonstrates an inadequate appreciation of the complexities in integrating cell, tissue, organ and systems during both acute disruptions in homeostasis by a single bout of exercise, and longer-term chronic adaptations to different types of exercise such as resistance and endurance. It is our opinion that those promoting drugs targeting a single or few molecules should not redefine the term 'exercise' and exercise concepts in an attempt to sensationalize findings. Additionally, the scientific criteria that the authors demand to be met to legitimately use the terms 'exercise pill' and 'exercise mimetic' are presented.

Linking performance and chronic disease risk: indices of physical performance are surrogates for health.

Recent studies have identified a remarkable association between indices of athletic performance and optimal health of the general public. Both high aerobic capacity and high skeletal muscle strength are associated with lower mortality. Furthermore, higher aerobic capacity and often higher skeletal muscle strength are associated with a lower prevalence of most chronic diseases. Also, maintenance of aerobic capacity and skeletal muscle strength by lifelong physical activity delays the biological ageing in most organ systems, therefore delaying premature death. These facts raise the question whether associations between high aerobic capacity and muscle strength are causally or associatively related to either metabolic health or elite performance. If a causal relationship was noted at the molecular level, it would have major public health implications. In this review, evidence is presented for the assertion that research on elite athletes and chronic disease prevention by exercise is actually addressing the same biochemical, physiological and genomic phenomena.

p21(Cip1) expression is increased in ambient oxygen, compared to estimated physiological (5%) levels in rat muscle precursor cell culture.

OBJECTIVE: While it is common practice to culture cells in the presence of ambient oxygen (approximately 21% O2), O2 level observed in the physiological environment is often much lower. Previous efforts to culture a variety of different stem cells, including muscle precursor cells (MPC), under O2 conditions that better mimic in vivo conditions have resulted in enhanced proliferation. In the present study, we hypothesized that 20% O2 in culture represents a sufficient stimulus to cause increased expression of two key negative regulators of the cell-cycle Cip/Kip family of cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), in MPCs. MATERIALS AND METHODS: MPCs were isolated from Fischer 344 x Brown Norway F(1) hybrid male rats and O2 was adjusted in culture using a tri-gas incubator. RESULTS: 5-Bromo-2'-deoxyuridine incorporation, cell number and nuclear proliferating cell nuclear antigen expression were all decreased after 48 h culture in 20% O2, compared to 5% O2. Twenty per cent O2 had no effect on either p27(Kip1) promoter activity or protein expression. Although p21(Cip1) promoter activity remained unchanged between 5% and 20% O2, there were significant increases in both p21(Cip1) mRNA and protein expression. Furthermore, 20% O2 caused an increase in p21(Cip1) mRNA stability and p53 transcription factor activity. CONCLUSION: These findings are considered important because they reveal p21(Cip1) as a critical regulatory protein that needs to be considered when interpreting proliferation data from MPCs studied in culture. In addition, O2-dependent regulation of MPC proliferation is relevant to conditions, including sarcopenia, heart failure, cancer and muscular dystrophy, where increased oxidative stress exists.

Changes in signalling molecule levels in 10-day hindlimb immobilized rat muscles.

AIM: Hindlimb immobilization produces similar percentage decreases in muscle mass in the predominantly type I soleus and type II vastus lateralis muscles. Consequently we hypothesized that the percentage changes in potential regulatory molecules for atrophy would be similar in the two muscle fibre types. METHODS: Therefore, the purpose of the current study was to measure phosphorylated p38 MAPK and JNK, as well as the protein levels of p53, growth arrest and DNA damage-inducible 45 (GADD45), and full-length poly(ADP-ribose) polymerase (PARP) to determine whether their changes in expression in the soleus and vastus lateralis muscles were similar at 10th day of hindlimb immobilization in young rats. RESULTS: Unexpectedly, preferential increases in phosphorylation of p38 MAPK and JNK and in the protein levels of p53, GADD45, as well as decreases in full-length PARP occurred in the soleus muscle, while only p38 phosphorylation increased in the white portion of the vastus lateralis muscle at 10th day of hindlimb immobilization. CONCLUSION: Taken together, these results are interpreted to suggest that some of regulatory processes or kinetics in the atrophy of type I and II muscle fibres during limb immobilization may differ at the 10th day of limb immobilization.

Late infantile onset krabbe disease in siblings with cortical degeneration and absence of cerebral globoid cells.

Krabbe disease, a disorder caused by the deficiency of lysosomal galactosylceramidase, is typically associated with cerebral white matter degeneration, cortical sparing, accumulation of macrophages ("globoid cells"), and ultrastructural needle-shaped inclusions. Two sisters presented with progressive neurological deterioration beginning before the age of 2.5 years. The first, who died at the age of 9 years, exhibited profound destruction of cerebral white matter with sparing of subcortical fibers but no globoid cells. The brain of the second, who died at the age of 15 years and who had a proven galactosylceramidase deficiency, exhibited white matter destruction, previously undescribed circumscribed spongiform cortical degeneration (postcentral, inferior temporal, cingulate), and cerebellar atrophy, but no globoid cells. The peripheral nerve biopsies from both girls exhibited typical needle-shaped inclusions in Schwann cells. These observations confirm the rare reports that Krabbe disease is not always associated with globoid cells in the brain. Psychosine, which accumulates in the brain, might be toxic to cortical neurons following prolonged survival. The reason for the regional susceptibility in the cerebral cortex is unknown.

Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages.

Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, approximately 10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to approximately 55%, while the population of CD34- or CD45-positive cells increased to approximately 30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.