Postoperative Severity Assessment in Sheep.

INTRODUCTION: Sheep are frequently used in translational surgical orthopedic studies. Naturally, a good pain management is mandatory for animal welfare, although it is also important with regard to data quality. However, methods for adequate severity assessment, especially considering pain, are rather rare regarding large animal models. Therefore, in the present study, accompanying a surgical pilot study, telemetry and the Sheep Grimace Scale (SGS) were used in addition to clinical scoring for severity assessment after surgical interventions in sheep. METHODS: Telemetric devices were implanted in a first surgery subcutaneously into four German black-headed mutton ewes (4-5 years, 77-115 kg). After 3-4 weeks of recovery, sheep underwent tendon ablation of the left M. infraspinatus. Clinical scoring and video recordings for SGS analysis were performed after both surgeries, and the heart rate (HR) and general activity were monitored by telemetry. RESULTS: Immediately after surgery, clinical score and HR were slightly increased, and activity was decreased in individual sheep after both surgeries. The SGS mildly elevated directly after transmitter implantation but increased to higher levels after tendon ablation immediately after surgery and on the following day. CONCLUSION: In summary, SGS- and telemetry-derived data were suitable to detect postoperative pain in sheep with the potential to improve individual pain recognition and postoperative management, which consequently contributes to refinement.

Increased DNA methylation of Dnmt3b targets impairs leukemogenesis.

The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

Human-directed behaviour in goats is not affected by short-term positive handling.

In addition to domestication, interactions with humans or task-specific training during ontogeny have been proposed to play a key role in explaining differences in human-animal communication across species. In livestock, even short-term positive interactions with caretakers or other reference persons can influence human-animal interaction at different levels and over different periods of time. In this study, we investigated human-directed behaviour in the 'unsolvable task' paradigm in two groups of domestic goats (Capra aegagrus hircus). One group was positively handled and habituated to a plastic box by the experimenter to retrieve a food reward, while the other group only received standard husbandry care and was habituated to the box without human assistance. In the unsolvable task, the lid was fixed to the box, with the reward inaccessible to the subjects. The goats were confronted with the unsolvable task three times. We observed no difference between the two groups regarding gaze and contact alternations with the experimenter when confronted with the task they cannot solve by themselves. The goats did not differ in their expression rates of both gaze and contact alternations over three repetitions of the unsolvable task; however, they showed earlier gaze and contact alternations in later trials. The results do not support the hypothesis that short-term positive handling or task-specific training by humans facilitates human-directed behaviour in goats. In contrast, standard husbandry care might be sufficient to establish humans as reference persons for farm animals in challenging situations.

Endocannabinoid administration affects taste preference and the expression of cannabinoid and opioid receptors in the amygdala of early lactating cows.

The aim of the study was to investigate the influence of intraperitoneal N-arachidonoylethanolamide (AEA) on taste preference for feed and water, tongue taste receptor signalling (TAS1R2, GNAT3), and endocannabinoid (CNR1, CNR2, GPR55) and opioid (OPRD1, OPRK1, OPRM1, OPRL1) receptors in the amygdala and nucleus accumbens in periparturient cows. We conducted taste preference tests using unaltered, umami-tasting, and sweet-tasting water and feed, before and after calving. After calving, eight cows received AEA injections (3 µg/(kg bodyweight × day), 25 days), whereas eight control (CON) cows received saline injections. Tissue was sampled 30 days after calving. Before calving, both cow groups preferred sweet-tasting feed and umami-tasting water. After calving, only the AEA-treated group preferred sweet-tasting feed, whereas the CON group showed no clear taste preference. In the amygdala, the mRNA expression of CNR1, OPRD1 (left hemisphere) and OPRK1 (right hemisphere) was lower in AEA animals than in CON animals, whereas no differences were found in the nucleus accumbens and tongue taste receptor expression. In conclusion, AEA administration enhanced existing taste preferences and reduced the expression of specific endocannabinoid and opioid receptors in the amygdala. The results support endocannabinoid-opioid interactions in the control of taste-dependent feed preference in early lactating cows.

Personality traits affect learning performance in dwarf goats (Capra hircus).

A wide range of species exhibit time- and context-consistent interindividual variation in a number of specific behaviors related to an individual's personality. Several studies have shown that individual differences in personality-associated behavioral traits have an impact on cognitive abilities. The aim of this study was to investigate the relationship between personality traits and learning abilities in dwarf goats. The behavior of 95 goats during a repeated open field (OF) and novel object test (NO) was analyzed, and two main components were identified using principal component analysis: boldness and activity. In parallel, the goats learned a 4-choice visual initial discrimination task (ID) and three subsequent reversal learning (RL) tasks. The number of animals that reached the learning criterion and the number of trials needed (TTC) in each task were calculated. Our results show that goats with the lowest learning performance in ID needed more TTC in RL1 and reached the learning criterion less frequently in RL2 and RL3 compared to animals with better learning performance in ID. This suggests a close relationship between initial learning and flexibility in learning behavior. To study the link between personality and learning, we conducted two analyses, one using only data from the first OF- and NO-test (momentary personality traits), while the other included both tests integrating only animals that were stable for their specific trait (stable personality traits). No relationship between personality and learning was found using data from only the first OF- and NO-test. However, stability in the trait boldness was found to have an effect on learning. Unbold goats outperformed bold goats in RL1. This finding supports the general hypothesis that bold animals tend to develop routines and show less flexibility in the context of learning than unbold individuals. Understanding how individual personality traits can affect cognitive abilities will help us gain insight into mechanisms that can constrain cognitive processing and adaptive behavioral responses.

The effect of age on discrimination learning and self-control in a marshmallow test for pigs.

Both humans and nonhuman animals need to show self-control and wait for a larger or better reward instead of a smaller or less preferred but instant reward on an everyday basis. We investigated whether this ability undergoes ontogenetic development in domestic pigs (similar to what is known in human infants) by testing if and for how long nine- and 16-week-old pigs wait for a larger amount of their preferred reward. In a delay-of-gratification task, animals first learned that a small reward was hidden under a white cup and a large reward under a black cup, and then the delay to deliver the large reward was gradually increased. The results show that older pigs could wait longer for a larger reward than younger pigs (10.6 ± 1.3 s vs. 5.2 ± 1.5 s), thereby confirming our hypothesis of ontogenetic development of self-control in pigs. This self-control is likely to be regulated by the behavioural inhibition system and associated systems. Self-control or, more specifically the lack of it may be involved in the development of abnormal behaviours, not only in humans but also in animals. Therefore, research on self-control in decision-making might provide a new perspective on abnormal behaviours in captive animals.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Coping Style Modifies General and Affective Autonomic Reactions of Domestic Pigs in Different Behavioral Contexts.

Based on individual adaptive strategies (coping), animals may react differently to environmental challenges in terms of behavior and physiology according to their emotional perception. Emotional valence as well as arousal may be derived by measuring vagal and sympathetic tone of the autonomic nervous system (ANS). We investigated the situation-dependent autonomic response of 16 domestic pigs with either a reactive or a proactive coping style, previously selected according to the backtest which is accepted in piglets to assess escape behavior. At 11 weeks of age, the pigs were equipped with an implantable telemetric device, and heart rate (HR), blood pressure (BP) and their respective variabilities (HRV, BPV) were recorded for 1 h daily over a time period of 10 days and analyzed in four behavioral contexts (resting, feeding, idling, handling). Additionally, the first minute of feeding and handling was used for a short-term analysis of these parameters in 10-s intervals. Data from day 1-3 (period 1) and day 8-10 (period 2) were grouped into two separate periods. Our results revealed general differences between the coping styles during feeding, resting and handling, with proactive pigs showing higher HR compared to reactive pigs. This elevated HR was based on either lower vagal (resting) or elevated sympathetic activation (feeding, handling). The short-term analysis of the autonomic activation during feeding revealed a physiological anticipation reaction in proactive pigs in period 1, whereas reactive pigs showed this reaction only in period 2. Food intake was characterized by sympathetic arousal with concurrent vagal withdrawal, which was more pronounced in proactive pigs. In contrast, neither coping style resulted in an anticipation reaction to handling. Vagal activation increased in reactive pigs during handling, while proactive pigs showed an increase in sympathetically driven arousal in period 2. Our findings confirm significant context-related differences in the general autonomic reaction of pigs with different coping styles. Additionally, the two coping styles differ in their affective appraisal over the time course of the experiment, underlining the importance of taking individual differences into account when studying affect and emotion in humans and animals.

Structural cartilage damage attracts circulating rheumatoid arthritis synovial fibroblasts into affected joints.

BACKGROUND: Rheumatoid arthritis synovial fibroblasts (RASFs) are known to travel via the bloodstream from sites of cartilage destruction to new locations where they reinitiate the destructive processes at distant articular cartilage surfaces. In this study, we examined the role of interleukin (IL)-1-induced cartilage changes and their chemotactic effect on RASF transmigratory capacity. METHODS: To investigate synovial fibroblast (SF) transmigration through endothelial layers, we used a modified Boyden chamber with an endothelioma cell layer (bEnd.5) as a barrier and IL-1-treated murine cartilage explants as a chemotactic stimulus for SFs from human tumor necrosis factor-transgenic (hTNFtg) mice. We injected recombinant IL-1 or collagenase into knee joints of wild-type mice, followed by tail vein injection of fluorescence-labeled hTNFtg SFs. The distribution and intensity of transmigrating hTNFtg SFs were measured by fluorescence reflectance imaging with X-ray coregistration. Toluidine blue staining was performed to evaluate the amount of cartilage destruction. RESULTS: Histomorphometric analyses and in vivo imaging revealed a high degree of cartilage proteoglycan loss after intra-articular IL-1 and collagenase injection, accompanied by an enhanced in vivo extravasation of hTNFtg SFs into the respective knee joints, suggesting that structural cartilage damage contributes significantly to the attraction of hTNFtg SFs into these joints. In vitro results showed that degraded cartilage was directly responsible for the enhanced transmigratory capacity because stimulation with IL-1-treated cartilage, but not with IL-1 or cartilage alone, was required to increase hTNFtg SF migration. CONCLUSIONS: The present data indicate that structural cartilage damage facilitates the migration of arthritic SF into affected joints. The prevention of early inflammatory cartilage damage may therefore help prevent the progression of rheumatoid arthritis and its spread to previously unaffected joints.

Interchangeability of Electrocardiography and Blood Pressure Measurement for Determining Heart Rate and Heart Rate Variability in Free-Moving Domestic Pigs in Various Behavioral Contexts.

This study assessed the interchangeability between heart rate (HR) and heart rate variability (HRV) measures derived from a series of interbeat intervals (IBIs) recorded via electrocardiogram (ECG) and intra-arterial blood pressure (BP) in various behavioral contexts. Five minutes of simultaneously recorded IBIs from ECG and BP signals in 11 female domestic pigs during resting, feeding, and active behavior were analyzed. Comparisons were made for measures of HR, the standard deviation of IBIs, and the root mean of the squared distances of subsequent IBIs derived from ECG and BP signals for each behavior category using statistical procedures with different explanatory power [linear regression, intraclass correlation coefficient (ICC), Bland and Altman plots, and analysis of variance (ANOVA)]. Linear regression showed a strong relationship for HR during all behaviors and for HRV during resting. Excellent ICCs [lower 95% confidence intervals (CI) >0.75] and narrow limits of agreement in all behavior categories were found for HR. ICCs for HRV reached the critical lower 95% CI value of 0.75 only during resting. Using Bland and Altman plots, HRV agreement was unacceptable for all of the behavior categories. ANOVA showed significant differences between the methods in terms of HRV. BP systematically overestimated HRV compared with ECG. Our findings reveal that HR data recorded via BP agree well those recorded using ECG independently of the activity of the subject, whereas ECG and BP cannot be used interchangeably in the context of HRV in free-moving domestic pigs.

Proteinase-Activated Receptor 1 (PAR1) regulates leukemic stem cell functions.

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.