Enrichment Devices Research Articles

Environmental Enrichment Devices Are Safe and Effective at Reducing Undesirable Behaviors in California Sea Lions and Northern Elephant Seals during Rehabilitation.

Environmental enrichment devices (EEDs) have been proven to promote positive wellbeing in zoos and aquariums, and support animals' reintroduction success; however, their use in rehabilitation centers is still limited. This pilot study investigated the safety and efficacy of three EEDs, Artificial Kelp, Horse KONG™ and Wubba Kong™, and their ability to decrease and/or eliminate undesired stereotypic behaviors or looking at staff/staff areas in seven wild California sea lions (CSLs) and eight northern elephant seals (NESs) undergoing rehabilitation in Southern California. Observers conducted instantaneous sampling once a minute during a 30-min baseline, followed by a 30-min EED implementation on one focal animal at a time. The data were analyzed with generalized linear mixed models (GLMM). All three EEDs were found to be durable and safe throughout the study. Our results show a significant reduction in stereotypical behaviors compared to no EED treatments in CSLs, with the most significant effect being with the Horse KONG™. The Wubba KONG™ and Artificial Kelp provisions also reduced the undesired behavior in both species of being focused on human caretakers. Individual preferences for specific EEDs were found within species and between species, with the NESs using EEDs more than the CSLs. This study highlights the beneficial aspect of EEDs for pinnipeds in rehabilitation centers for improving their quality of life.

Laser environmental enrichment and Spirulina algae moderately improve Ross 708 broiler performance without reducing welfare

Modern commercial broilersare genetically selected for increased growth rate, improved feed efficiency,and a significant proportion of breast muscle tissue. Reducing feed conversionrate and days to market weight are of key interest to the industry from asustainability perspective. Dietary and environmental interventions that canattain these improvements without negatively impacting animal welfare are beingincreasingly studied and implemented. In the current study, a previously testedlaser enrichment device and a Spirulina algae feed additive were introduced to 600Ross 708 broilers grown for 7 weeks. Laser enrichment and dietary algae inclusioneach increased weight gain during the grower period (P<0.05 and P<0.10,respectively). Laser enrichment alone increased body weight at d28 by 77g and ond42 by 114g compared to CON (no laser enrichment; P<0.05). Neitherenrichment nor algae inclusion impacted litter moisture or increased contactdermatitis incidence, indicating a performance benefit without reducinganimal-based welfare outcomes.

Influence of an Automatic Enrichment Device on Laying Hen Behavior and Plumage Condition.

Feather pecking and cannibalism are prominent problems in modern laying hen husbandry. Among the various approaches to address this issue, environmental enrichment plays a crucial role. In this on-farm study, four winter gardens of an organic farm henhouse were equipped with an automatic enrichment device. Different quantities of downpipes dosing grain on rough-coated pecking plates (PPs) were tested. One group served as a control (CG) without an automatic enrichment device, while the others were offered different numbers of PPs, with one equipped with a doubled amount of PPs (DEG) compared to the other two groups (SEG). Video analyses of the hens' duration of stay and behaviors in the recorded winter garden area and around the PPs were performed, and regular assessments of the plumage condition were conducted. By the end of the production cycle, no hens with intact plumage were found, with hens in the CG and DEG showing worse scores and earlier deterioration in the plumage condition than in the SEG. The offer of PPs showed a significant influence on the duration of stay in the filmed area. Hens in the DEG stayed significantly longer (mean: 129 s, SD: 126 s) than those in the CG (mean: 79 s, SD: 91 s; p < 0.05) and SEG (mean: 75 s, SD: 83 s; p < 0.005). On the performed behavioral bouts per hen and minute (CG mean bouts/minute (SD): 5.47 (2.92); SEG mean (SD): 5.33 (2.76); SEG mean (SD): 5.81 (3.24)), no significant influences were detected. Environmental pecking was the behavior most frequently observed in all winter gardens, where, particularly around the PPs, pecking at the device was observed. Therefore, the enrichment device can be assessed as well accepted by the hens in winter gardens. The effect of the device on the plumage condition remains unclear, with external factors probably showing a greater influence than the enrichment.

RNase H2 triggered visual loop-mediated isothermal amplification combining smartphone assisted all-in-one aptamer magnetic enrichment device for ultrasensitive culture-independent detection of Salmonella Typhimurium in chicken meat

Pre-enrichment cultural continues to remain time-consuming and labor-intensive in the detection of low-concentration bacteria in meat samples. Culture-independent detection method is urgently needed in POCT technology. In order to be able to proceed with culture-independent pre-enrichment for low-concentration pathogen detection, a RNase H2 enzyme triggered all-in-one device-based visual Loop-mediated Isothermal Amplification detection method was developed, combining with Salmonella Typhimurium specific Aptamer Magnetic Enrichment steps, termed as the AME-LAMP method. In order to better enable the AME-LAMP method to perform on-site culture-independent detection, a palm-held all-in-one device (PAD) was designed and manufactured. Further, a self-developed smart phone fluorescence analysis app could complete end-point detection, thus eliminating miscalculation. RNase H2 Enzyme triggered AME-LAMP method could detect Salmonella as low as 5.5 CFU/mL while conventional LAMP only detected 550 CFU/mL. By verifying the detection performance of AME-LAMP methods in chicken meat samples, AME-LAMP shows the potential to achieve ultrasensitive Salmonella detection by comparing with ISO isolation method and normal LAMP. With the assistance of smart phone-based PAD, it can be reliably applied for the on-site culture-independent detection of Salmonella Typhimurium.

All-in-One Preparation Strategy Integrated in a Miniaturized Device for Fast Analyses of Biomarkers in Biofluids by Surface Enhanced Raman Scattering.

Complex and tedious sample preparation processes have greatly limited rapid analyses of biological samples. In this work, an all-in-one sample preparation strategy based on a miniaturized gas membrane separation/oven ring enrichment (GMS/ORE) device was developed for efficient surface enhanced Raman scattering (SERS) analyses of trace biomarkers in biofluid samples. This strategy integrating gasification separation, liquid trapping, derivatization SERS activation, and coffee-ring enrichment could highly promote the efficiency of sample preparation. Meanwhile, the edges of membranes modified by the hydrophobic-infusing slippery liquid-induced uniform "coffee-ring" effect could significantly improve the sensitivity and stability for SERS quantification. By adapting proper derivatization approaches to the miniaturized GMS/ORE pretreatment, the matrix effects in samples could be prominently eliminated, and clear SERS responses could be obtained for the selective analyses of target biomarkers. The miniaturized GMS/ORE device was practically applied for SERS analyses of trace biomarkers in biofluids, including hydrogen sulfide in saliva samples, creatinine in serum samples, and sarcosine, creatinine, and dimethyl disulfide in urine samples. Accurate quantification of all biomarkers was achieved with recoveries of 89.5%-120.0%, and the contents found by GMS/ORE-SERS matched well with those found by corresponding chromatographic methods with relative errors from -8.6% to 9.3%. The miniaturized GMS/ORE device with multiple parallel processing units could simultaneously treat eight samples in one run with a total analysis time of 40 min. Such an efficient all-in-one strategy integrated on a miniaturized device possesses great potential for fast on-site/point-of-care detection in analytical science and clinical medicine.

Cetacean responses to violation of expectation paradigm in a free-swim context.

The investigation of individual responses to unexpected stimuli or outcomes provides insights into basic cognitive processes, such as mental representations, emotional states of surprise, and detections of anomalies. Three experiments using a violation of expectation paradigm were conducted with 12 belugas and 17 bottlenose dolphins in managed care to test two classes of stimuli (humans and objects) in manipulated sequences of familiar and unfamiliar humans (Study 1, trainers and strangers), familiar and unfamiliar objects (Study 2, typical enrichment devices and new objects), and finally objects and humans (Study 3). Gaze durations were assessed for each condition in a given study during free-swim contexts. The results supported previous findings that visual stimuli, regardless of class, were stimulating and intriguing for both belugas and bottlenose dolphins. Belugas were more likely to gaze longer at human and object stimuli and tended to gaze longer at unexpected experiences than control or expected experiences. Bottlenose dolphins showed similar trends except when objects were involved. Individual variability was present for both species with some individuals showing stronger patterns of responses for expected experiences than others. After 2years of intermittent experiments, belugas and bottlenose dolphins in managed care maintained their curiosity about visual stimuli, for which they received no primary reinforcement. Investigating responses to unexpected stimuli with animals in managed care may provide insight into how these animals respond to biologically relevant conditions, such as boat presence, predators, and unfamiliar conspecifics.

A Chelating Resin of EDTA-Type Modified Cross-Linking Polystyrene to Preconcentrate Trace Metals for Seawater Detection.

The determination of trace metals in seawater is an important project of marine environmental monitoring. However, the presence of many alkali metal ions with high concentration, such as sodium ion, seriously interferes with the detection limit and accuracy of atomic absorption spectrometry (AAS, flame/graphite furnace integrated). The conventional chemical methods for the enrichment of trace metals are complex, and low boiling point organic solvents are used. In this paper, a kind of commercial cross-linked polystyrene resin microspheres was chloromethylated and aminated to introduce EDTA-type amino polycarboxylic groups and then loaded in a column as the absorption filler. A set of seawater pretreatment and enrichment devices was designed and assembled. The enriching process and conditions of trace Cu, Zn, Pb, and Cd in standard seawater were studied. 10 g of the modified resin could enrich the equivalent seawater and remove successfully the light metal ions. pH = 5∼9 and 0.2 mL/min of the flow rate were the suitable conditions for preconcentration. The enriched metal ions in the eluent were analyzed on the AAS. Compared with the conventional solvent method, the novel material and enrichment device have high preconcentration efficiency, strong anti-interference ability, and low cost and could be directly applied for routine seawater detection.

Multivariate analysis of liquid biopsies for real-time detection of patients with biofilm-associated infections (BAI)

Biofilm-associated infections (BAI) are chronic infections that are refractory to standard antibiotic therapy and challenging to diagnose. Detecting biofilms in patients remains a major challenge in the clinical field. Here, we introduced a microfluidic-based label-free and multivariate analysis (LF-MA) platform to assess biofilm-associated infection disease via multivariate analysis of severity parameters for point-of-care (POC) management. The integrated LF-MA platform consisted of two components: Biofilm Enrichment Device (BED) and Severity Detection Device (SDD), and allowed simultaneous real-time biofilm enrichment and viscosity-based BAI severity detection. High recovery efficiencies (>80 %) were observed for both gram-positive and gram-negative strains. A novel biosensor HMS indicator (Healthy: 1–3+, Mild: 1–3-, Severe: 1 + 3-) was developed to evaluate the severity of BAI based on microbeads distribution in target outletsSDD (for viscosity assessment) and the presence of biofilms. The one-step strategy was validated with patient-derived clinical isolates and could be completed within 2 h. We envisioned that the ease of operations and derived HMS biosensor indicator could facilitate new patient-centric approaches for rapid and multivariate assessment of patients with BAI.

An Evaluation of Ethograms Measuring Distinct Features of Enrichment Use by Captive Chimpanzees (Pan troglodytes).

Simple SummaryEnvironmental enrichment for chimpanzees is important in order to minimize boredom and stress in captivity and to provide opportunities to engage in species-typical behaviors. However, few studies have investigated potential associations between enrichment objects, manipulation behaviors, and social contexts, nor have they examined if individual chimpanzees vary in their enrichment object preferences. In the current study, three ethograms were used to code the use of enrichment objects, engagement in manipulation behaviors, and social contexts of enrichment use of captive chimpanzees (Pan troglodytes). Data mining from a video archive consisting of 2054 videos collected over a decade allowed the ethograms to be applied to 732 min and 58 s of videos. Some enrichment objects were more often associated with specific manipulation behaviors and social contexts, indicating that enrichment objects might serve distinct social and behavioral purposes. The chimpanzees differed in their enrichment object preferences, suggesting that caregivers of captive chimpanzees should consider individual needs when providing enrichment in order to improve chimpanzees’ experiences in captivity. Finally, the majority of enrichment object use and manipulation behaviors were able to be categorized, indicating that our ethograms were largely effective in coding enrichment use.Environmental enrichment provides mental stimulation and minimizes abnormal behaviors in captive animals. In captive chimpanzees, individual animals may vary in the ways in which they benefit from enrichment or use enrichment devices, so investigating nuances in enrichment use may improve the welfare of captive chimpanzees. In the current study, three ethograms measuring distinct features of enrichment use (i.e., enrichment object, manipulation behavior, and social context) were evaluated by coding videos of captive chimpanzees (Pan troglodytes) at Chimpanzee Sanctuary Northwest in Cle Elum, WA. A total of 732 min and 58 s of video footage was coded from a larger video archive (i.e., 2054 videos) of enrichment use that spanned a decade. A principal component analysis (PCA) revealed that different categories of enrichment objects were more often associated with specific manipulation behaviors and social contexts, suggesting that enrichment objects might fulfill different behavioral and social needs in captivity. Specifically, toy objects were associated with active tactile behaviors in affiliative contexts while oral behaviors were used with foraging objects in solitary contexts. Additionally, individual chimpanzees showed unique preferences for enrichment objects, indicating that caregivers of captive chimpanzees should consider individual needs instead of a “one size fits all” approach to enrichment provisions.

O056 Achieving optimal clinical outcomes in autologous fat grafting (AFG): a systematic review of processing techniques

Abstract Introduction AFG is an increasingly popular technique in reconstructive and cosmetic surgery but significant variability in long-term graft retention continues to be a limitation. This results in unreliable clinical outcomes and revision procedures. There is no consensus on the optimal methodology of graft processing. This systematic review identifies the latest evidence base supporting different processing techniques. Methods A systematic literature search was conducted using PubMed, SCOPUS, and the Cochrane electronic databases. Studies comparing AFG processing techniques and reporting long-term patient outcomes (≥3 months) were identified. Data collected included the technical aspects of techniques, cohort characteristics, complication rates, volume retention. Systematic assessment of study quality was also performed. Results Twenty-four studies (2928 patients) providing at least level three evidence were included. Processing techniques evaluated included centrifugation, decantation, washing, filtration, gauze rolling, as well as commercial devices and ASC enrichment methods. There was variable reporting of complications and volume retention rates. Complications were infrequent; palpable cysts (0–18%), cellulitis (0–8%) and fat necrosis (0–36.4%) were the most commonly reported. No significant differences in long-term volume retention between techniques were found in AFG in the breast. In head and neck patients, greater volume retention were documented in ASC enrichment (64.8–95.8%) and commercial devices (41.2%) compared to centrifugation (31.8–76%). Conclusion The quality of AFG research has improved significantly in recent years but has heterogeneity in reporting outcomes. ASC (adipose derived stem/stromal cells) enrichment methods and commercial devices results in superior long-term volume retention in facial but not breast fat grafting. Take-home message Adipose derived stem/stromal cell enrichment methods and commercial devices have superior long term volume retention in facial but not breast autologous fat grafting [level of evidence II]. Randomised control trials are necessary to identify if certain processing methods are superior to others in volume retention and complication rates.

Isotopic Enriched and Natural SiC Junction Barrier Schottky Diodes Under Heavy Ion Irradiation

The radiation tolerance of isotopic enriched and natural silicon carbide junction barrier Schottky diodes are compared under heavy ion irradiation. Both types of devices experience leakage current degradation as well as single-event burnout events. The results were comparable, although the data may indicate a marginally lower thresholds for the isotopic enriched devices at lower linear energy transfer (LET). Slightly higher reverse bias threshold values for leakage current degradation were also observed compared to previously published work.

Evaluating Cognitive Enrichment for Zoo-Housed Gorillas Using Facial Recognition.

The use of computer technology within zoos is becoming increasingly popular to help achieve high animal welfare standards. However, despite its various positive applications to wildlife in recent years, there has been little uptake of machine learning in zoo animal care. In this paper, we describe how a facial recognition system, developed using machine learning, was embedded within a cognitive enrichment device (a vertical, modular finger maze) for a troop of seven Western lowland gorillas (Gorilla gorilla gorilla) at Bristol Zoo Gardens, UK. We explored whether machine learning could automatically identify individual gorillas through facial recognition, and automate the collection of device-use data including the order, frequency and duration of use by the troop. Concurrent traditional video recording and behavioral coding by eye was undertaken for comparison. The facial recognition system was very effective at identifying individual gorillas (97% mean average precision) and could automate specific downstream tasks (for example, duration of engagement). However, its development was a heavy investment, requiring specialized hardware and interdisciplinary expertise. Therefore, we suggest a system like this is only appropriate for long-term projects. Additionally, researcher input was still required to visually identify which maze modules were being used by gorillas and how. This highlights the need for additional technology, such as infrared sensors, to fully automate cognitive enrichment evaluation. To end, we describe a future system that combines machine learning and sensor technology which could automate the collection of data in real-time for use by researchers and animal care staff.

A review of welfare indicators for sea turtles undergoing rehabilitation, with emphasis on environmental enrichment

AbstractFor animals undergoing rehabilitation it is vital to monitor welfare in a way that is feasible, practical, and limits stress to the animal. The industry gold standard is to assess welfare under the Five Domains model, including nutrition, environment, physical health, and behaviour as the first four physical domains and mental domain as the fifth. Feasibility and effectiveness of these domains for assessing welfare of sea turtles undergoing rehabilitation were reviewed and it was determined that the mental state can be best assessed through behavioural changes. A scoping review of the literature was conducted using Scopus and Web of Science to investigate use of environmental enrichment devices (EEDs) as a measure of welfare in sea turtles. Behavioural assessments using EEDs were found to be well-documented; however, most EED studies pertained largely to livestock or zoo animals. Furthermore, studies rarely concentrated on reptiles, and specifically sea turtles. Results also showed that certain welfare assessment methods may be less appropriate for short-term captivity experienced during rehabilitation. Additionally, the hospital environment limits the ability to address some of the domains (ie biosecurity, feasibility, safety of turtle, etc, might be compromised). This review shows that only three of the nine environmental enrichment strategies described in the literature suit the specific requirements of sea turtles in rehabilitation: feeding, tactile, and structural. It is documented that turtles display behaviours that would benefit from EEDs and, therefore, more specific studies are needed to ensure the best welfare outcomes for sea turtles undergoing rehabilitation.

Electrodialytic Enrichment and Matrix Conversion for the Determination of Trace Metals in Ultra-Pure Water.

The presence of trace contaminants in ultra-pure water (UPW) used in fabrication process can greatly affect the yield and quality of industrial products. In the present study, the electrodialytic enrichment of metal cations as a means of continuously monitoring the UPW quality was studied. A newly designed electrodialytic enrichment device (EED) was used to quantitatively transfer metal ions from samples to dilute nitric acid, which was then directly introduced into an inductively coupled plasma—mass spectrometry (ICP–MS) instrument. This process could be performed without contamination of the sample, and the enrichment factor was solely dependent on the flow rate ratio of the sample and acceptor solutions. The transference of analytes into the acidic solution improved the responsivity of the ICP–MS analysis, especially at low concentrations of less than 1 μg/L. Blank solutions to support the analysis of UPW could be produced using the EED effluent, from which metal ions were quantitatively removed. In addition, calibration curves with concentration ranges of several nanograms per liter were obtained by preparing standards using a dynamic gravimetric method while employing a single bottle and continuous mass monitoring to avoid any contamination from the volumetric flasks. The sensitivities associated with the ICP–MS analysis of a number of trace metal ions were improved by one or two orders of magnitude. The data show that the present EED is able to continuously produce enriched analyte solutions to allow the ongoing monitoring of UPW quality.

A Place to Be? Use of Winter Gardens with and without Automatic Enrichment Devices by Laying Hens

Since the abolition of beak trimming in laying hens in Germany, the importance of adequate enrichment material to help reduce feather pecking and cannibalism has grown. Here we tested an automatic enrichment device dosing grain via rough-coated pecking plates (PPs) on an organic farm, comparing its offer in four winter gardens (WGs). Winter garden (WG) 1 served as the control area without an automatic enrichment device, while WGs 2–4 offered different quantities of PPs, with WG 2 offering double the amount of PPs as WGs 3 and 4. The number of laying hens (Lohmann Brown Lite) per m² and close to the enrichment device (one hen‘s body length around) was determined using photo records. The usage behavior of the hens differed in the four WGs and with the animals’ age. Over the whole husbandry period (60 weeks), on average, 1.48 hens/m² were detected in the control area (WG 1), and a mean of 2.27 hens/m² in the enriched WGs. Compared to WG 1, the number of hens per m² was higher in WG 2 (2.43 hens/m²) and WG 3 (2.59 hens/m²) (p &lt; 0.05), but similar in WG 4 (1.79 hens/m²). At the end of the husbandry period, fewer animals (mean of all WGs: 1.43 hens/m²) used the WGs than from beginning to the middle of the laying period (mean of all WGs: 2.05–2.15 hens/m²; p &lt; 0.05). Our data indicate that the automatic enrichment device positively influences the animals’ use of the WG.

Study of green surface-enhanced Raman spectroscopy determination of methanol in alcoholic beverages

There are two main challenges in completing surface-enhanced Raman spectroscopy (SERS) detection. One is the small Raman scattering cross-section of methanol, and the other is the derivatization strategy. They were solved by coupling the catalytic strategy with a thermal purge separation and enrichment device. In this study, quantitative analysis of methanol in alcoholic beverages was performed as methanol was catalyzed to formaldehyde. For detecting methanol in low-alcohol beverages, the acetaldehyde derivatives generated by the interaction of ethanol-catalyzed oxide acetaldehyde with 3-methyl 2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH) during the catalytic process will not cause large interference with the SERS signal of formaldehyde. In this case, MBTH with higher sensitivity is selected for the derivatization operation. In the analysis of methanol in high-alcohol beverages, acetylacetonate derivatization without interaction with acetaldehyde was used to avoid the interference of the acetaldehyde generated from ethanol. Based on the detection of formaldehyde in methanol standard solutions, methanol was detected in wine and sake as well as in high-alcohol beverages. Thus, a convenient and rapid method was achieved for the detection of methanol in alcoholic beverages.

Testing use of the first multi-partner cognitive enrichment devices by a group of male bottlenose dolphins.

Alliance formation plays a crucial part in male dolphins' lives. These partnerships may last for decades or even for a lifetime; thus, partner choice and the maintenance of these relationships are both considered key components of alliance formation. In our previous investigations, pairs of adult male dolphins showed a high success rate in cooperative manipulation of a cognitive enrichment device. Here, we introduced two novel cognitive enrichment devices to the group of five dolphins, facilitating simultaneous actions for not only pairs, but for three or even four dolphins. The devices were made of PVC tubes, fittings and caps equipped with rope handles, creating a three-way (T-shape) and a four-way (TT-shape) device. The devices were filled with fish and ice and were designed to be opened by simultaneous pull of the handles. Both devices were tested in 12 trials (each lasted for 15min), separately. Only one of the caps could be opened, the others were affixed with the position of the openable cap counter-balanced over the trials. Although the dolphins received no training regarding the manipulation of the devices, they were successful in cooperatively opening the three-way devices in 10/12 of trials (70% by two and 30% by three dolphins) and the four-way devices also in 10/12 trials (50% by two, 40% by three and 10% by four dolphins). The dolphins interacted with the devices during the entire testing time, and this was mostly spent in cooperative play (77% and 56% of the test duration with the three-way and four-way device, respectively). The majority of the cooperative play was observed between one particular pair of dolphins that was temporarily associated with a third or sometimes even with a fourth dolphin. These findings demonstrate the first successful use of multi-partner cooperative enrichment devices, providing information on the social organization of a male dolphin group.

Effects of Complex Feeding Enrichment on the Behavior of Captive Malayan Sun Bears (Helarctos malayanus)

ABSTRACT All zoos grapple with challenges of keeping captive animals engaged in natural behaviors, especially for bears which prove to be among the more challenging species to keep stimulated. In captivity, a common indicator of poor welfare is the presence of stereotypic behaviors. This study tests whether providing complex feeding enrichment devices decreases the duration of stereotypic behavior and increases enrichment interaction for three adult female sun bears (Helarctos malayanus) at Oakland Zoo in California. This study uses two different enrichment devices presented at three complexity levels. After three weeks of baseline data collection when no complex enrichment is present, the complex enrichment is introduced three times a week per level over six weeks. Sun bear interaction with the enrichment devices is also measured to examine the effect of complexity on enrichment use. Providing complex enrichment decreased the duration of stereotypic behavior when compared to baseline. Across the six weeks, the duration of stereotypic behavior is significantly less on the complex enrichment days compared to non-complex enrichment days. The complex enrichment has variable effects on enrichment use. These results suggest that providing complex enrichment may have a positive influence on the behavior of captive bears.

Laser Enrichment Device Stimulates Broiler Laser-Following Behavior While Increasing Individual Bird Locomotion and Pen-Wide Movement

Genetic selection for fast growth rate and high breast muscle yield in modern broilers has unintended effects on animal welfare and behavior, namely in terms of inactivity and leg disorders. We hypothesized that exercise stimulated through environmental enrichment could positively stimulate pen-wide activity and improve bird welfare. The study objectives were to implement a laser enrichment device to motivate active and feeding behaviors throughout the pen. Twelve hundred Ross 708 broilers were randomly assigned to enrichment (LASER; laser enrichment, or CON; no laser enrichment) for 49 d. Seventy focal birds were randomly assigned to 14 video-recorded pens for behavioral analysis, including focal bird home pen behavior and walking distance. Pen-wide activity was also measured during the 4-min laser periods, four times daily, d0–8, and 1 day weekly, wk 1–6. Focal birds were gait scored wk 1–6, and were euthanized on d42 for tibia bone mineral content, density, and bone breaking strength analysis. Time spent active was increased in LASER-enriched birds compared to CON on wk 3–5 by up to 214% (wk 4), and percent of time at the feeder was increased in LASER-enriched birds by 761% on wk 4 (P&amp;lt; 0.05). Peak percent of birds following the laser (LASER-enriched pens only) was observed on d0 (8.52%). Over wk 1–6, peak laser-following behavior was observed on wk 3 (3.07% of birds). Percent of birds moving during laser periods was increased in LASER-enriched pens on d0, 1, 2, 6, 7, and 8, with a percent increase of 68.7% observed on d1 (P&amp;lt; 0.05). Percent of birds moving (laser-following or not) was increased on wk 1, 3, and 4 in LASER-enriched pens, with an increase of 69.7% observed on wk 4 (P&amp;lt; 0.05). No differences were found in tibia measures. These data indicate that laser enrichment stimulated voluntary locomotion through wk 5 and laser-following behavior through wk 6, and that the relatively small percent of birds actively following the laser stimulated pen-wide movement above the level of the CON through wk 4 on study.

Immediate Sample Fixation Increases Circulating Tumour Cell (CTC) Capture and Preserves Phenotype in Head and Neck Squamous Cell Carcinoma: Towards a Standardised Approach to Microfluidic CTC Biomarker Discovery.

Simple SummaryCirculating tumour cells (CTCs) have shown potential to act as markers of disease and prognosis in head and neck squamous cell carcinoma (HNSCC). However, there are a number of methods and devices reported to isolate and characterise CTCs. Translating CTC markers to clinical practice, for patient benefit, requires a reliable, reproducible and standardised approach. We report the benefit of the Parsortix microfluidic CTC enrichment platform in HNSCC. We demonstrate consistent cell capture rates between 10 and 100 cells/mL of whole blood. Analysis of gene expression with unfixed cells before and after Parsortix enrichment demonstrated a cell stress response and downregulation of key genes. We highlight the benefit of using a fixative blood collection tube (Transfix) to increase cell capture rate and preserve the CTC marker expression profile. Such evidence is crucial when designing sample processing protocols for large cohort multi-centre clinical trials investigating CTCs in any cancer type.Introduction: Research demonstrates strong evidence that circulating tumour cells (CTCs) can provide diagnostic and/or prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC) and a potential tool for therapeutic stratification. However, the question still remains as to the optimum method of CTC enrichment and how this can be translated into clinical practice. We aimed to evaluate the Parsortix microfluidic device for CTC enrichment and characterisation in HNSCC, seeking to optimise a sample collection and processing protocol that preserves CTC integrity and phenotype. Method: Spiking experiments of the FaDu and SCC040 HNSCC cell lines were used to determine the Parsortix capture rate of rare “CTC-like” cells. Capture rates of cancer cells spiked into EDTA blood collections tubes (BCTs) were compared to the Transfix fixative BCT and Cytodelics whole blood freezing protocol. The Lexogen Quantseq library preparation was used to profile gene expression of unfixed cells before and after microfluidic enrichment and enriched cell line spiked Transfix blood samples. An antibody panel was optimised to enable immunofluorescence microscopy CTC detection in HNSCC patient Transfix blood samples, using epithelial (EpCAM) and mesenchymal (N-cadherin) CTC markers. Results: Across a spiked cell concentration range of 9–129 cells/mL, Parsortix demonstrated a mean cell capture rate of 53.5% for unfixed cells, with no significant relationship between spiked cell concentration and capture rate. Samples preserved in Transfix BCTs demonstrated significantly increased capture rates at 0 h (time to processing) compared to EDTA BCTs (65.3% vs. 51.0%). Capture rates in Transfix BCTs were maintained at 24 h and 72 h timepoints, but dropped significantly in EDTA BCTs. Gene expression profiling revealed that microfluidic enrichment of unfixed cell lines caused downregulation of RNA processing/binding gene pathways and upregulation of genes involved in cell injury, apoptosis and oxidative stress. RNA was successfully extracted and sequenced from Transfix preserved cells enriched using Parsortix, demonstrating epithelial specific transcripts from spiked cells. In a proof-of-concept cohort of four patients with advanced HNSCC, CTCs were successfully identified and visualised with epithelial and epithelial-mesenchymal phenotypes. Conclusion: We have optimised a protocol for detection of CTCs in HNSCC with the Parsortix microfluidic device, using Transfix BCTs. We report a significant benefit, both in terms of cell capture rates and preserving cell phenotype, for using a fixative BCT- particularly if samples are stored before processing. In the design of large cohort multi-site clinical trials, such data are of paramount importance.