Environmental Complexity and Reduced Stocking Density Promote Positive Behavioral Outcomes in Broiler Chickens.

The objective was to evaluate the impacts of a complex environment and stocking density on Ross 708 broiler chicken behaviors. Eight pens contained either high complexity (HC) or low complexity (LC) environments, and high (HD) or low (LD) density. Through focal-animal sampling, the frequency and duration of behaviors were recorded continuously for 5 min at two timepoints for one day in weeks 2, 4, and 7. Birds were active for 30% of the observed time, with birds showing more activity in HC compared with LC. Birds in HC pens spent more time preening and foraging than birds in LC pens, which was interpreted as a positive outcome. Dustbathing and play were not impacted by complexity, possibly due to the observation method. Birds were more frequently active at HD compared with LD, but did not spend more time being active, suggesting disturbances. Birds foraged, drank, and ate less frequently in HD compared with LD, presumably because birds had more difficulty accessing resources. Activity and active behaviors reduced as birds aged, while preening frequency increased, possibly due to frustration, but this was not confirmed. Perching was unaffected by age, showing a persistent motivation to perform the behavior. Our results indicate that a complex environment provides positive stimulation for foraging, locomotion, preening, and overall activity. Despite reduced activity, many benefits of the tested environmental complexity and low density persisted as birds aged.

Measuring Chronic Stress in Broiler Chickens: Effects of Environmental Complexity and Stocking Density on Immunoglobulin-A Levels.

Commercial housing conditions may contribute to chronic negative stress in broiler chickens, reducing their animal welfare. The objective of this study was to determine how secretory (fecal) and plasma immunoglobulin-A (IgA) levels in fast-growing broilers respond to positive and negative housing conditions. In three replicated experiments, male Ross 708 broilers (n = 1650/experiment) were housed in a 2 × 2 factorial study of high or low environmental complexity and high or low stocking density. In experiments 1 and 3 but not in experiment 2, high complexity tended to positively impact day 48 plasma IgA concentrations. When three experiments were combined, high complexity positively impacted day 48 plasma IgA concentrations. Stocking density and the complexity × density interaction did not impact day 48 plasma IgA concentrations. Environmental complexity and the complexity × density interaction did not impact day 48 secretory IgA concentrations. A high stocking density negatively impacted day 48 secretory IgA concentrations overall but not in individual experiments. These results suggest that environmental complexity decreased chronic stress, while a high stocking density increased chronic stress. Thus, plasma IgA levels increased under high-complexity housing conditions (at day 48), and secretory IgA levels (at day 48) decreased under high-density conditions, suggesting that chronic stress differed among treatments. Therefore, these measures may be useful for quantifying chronic stress but only if the statistical power is high. Future research should replicate these findings under similar and different housing conditions to confirm the suitability of IgA as a measure of chronic stress in broiler chickens.

Effects of Housing System on Anxiety, Chronic Stress, Fear, and Immune Function in Bovan Brown Laying Hens.

The scientific community needs objective measures to appropriately assess animal welfare. The study objective was to assess the impact of housing system on novel physiological and behavioral measurements of animal welfare for laying hens, including secretory and plasma Immunoglobulin (IgA; immune function), feather corticosterone (chronic stress), and attention bias testing (ABT; anxiety), in addition to the well-validated tonic immobility test (TI; fearfulness). To test this, 184 Bovan brown hens were housed in 28 conventional cages (3 birds/cage) and 4 enriched pens (25 birds/pen). Feces, blood, and feathers were collected 4 times between week 22 and 43 to quantify secretory and plasma IgA and feather corticosterone concentrations. TI tests and ABT were performed once. Hens that were from cages tended to show longer TI, had increased feather corticosterone, and decreased secretory IgA at 22 weeks of age. The caged hens fed quicker, and more hens fed during the ABT compared to the penned hens. Hens that were in conventional cages showed somewhat poorer welfare outcomes than the hens in enriched pens, as indicated by increased chronic stress, decreased immune function at 22 weeks of age but no other ages, somewhat increased fear, but reduced anxiety. Overall, these novel markers show some appropriate contrast between housing treatments and may be useful in an animal welfare assessment context for laying hens. More research is needed to confirm these findings.

Effect of Environmental Complexity and Stocking Density on Fear and Anxiety in Broiler Chickens.

Barren housing and high stocking densities may contribute to negative affective states in broiler chickens, reducing their welfare. We investigated the effects of environmental complexity and stocking density on broilers' attention bias (measure of anxiety) and tonic immobility (measure of fear). In Experiment 1, individual birds were tested for attention bias (n = 60) and in Experiment 2, groups of three birds were tested (n = 144). Tonic immobility testing was performed on days 12 and 26 (n = 36) in Experiment 1, and on day 19 (n = 72) in Experiment 2. In Experiment 1, no differences were observed in the attention bias test. In Experiment 2, birds from high-complexity pens began feeding faster and more birds resumed feeding than from low-complexity pens following playback of an alarm call, suggesting that birds housed in the complex environment were less anxious. Furthermore, birds housed in high-density or high-complexity pens had shorter tonic immobility durations on day 12 compared to day 26 in Experiment 1. In Experiment 2, birds from high-density pens had shorter tonic immobility durations than birds housed in low-density pens, which is contrary to expectations. Our results suggest that birds at 3 weeks of age were less fearful under high stocking density conditions than low density conditions. In addition, results indicated that the complex environment improved welfare of broilers through reduced anxiety.

Intimate Partner Violence and Pet Abuse: Responding Law Enforcement Officers' Observations and Victim Reports From the Scene.

The risk of harm/injury in homes where intimate partner violence (IPV) occurs is not limited to humans; animals reside in as many as 80% of these homes and may be at substantial risk of suffering severe or fatal injury. Gaining a better understanding of IPV-pet abuse overlap is imperative in more accurately identifying the risks of harm for all individuals and animals residing in these homes. The objectives of this study were to utilize law enforcement officers' observations and IPV victim reports from the scene of the incident to (a) determine the prevalence of pet abuse perpetration among suspects involved in IPV incidents, (b) compare characteristics of IPV incidents and the home environments in which they occur when the suspect has a history of pet abuse with incidents involving suspects with no reported history of pet abuse, and (c) compare IPV incident outcomes involving suspects with a history of pet abuse with those involving suspects with no reported history of pet abuse. IPV victims residing in homes with a suspect who has a history of pet abuse often describe "extremely high-risk" environments. With nearly 80% reporting concern that they will eventually be killed by the suspect, victims in these environments should be considered at significant risk of suffering serious injury or death. In addition, IPV victims involved in incidents with a suspect that has a history of pet abuse were significantly more likely to have had at least one prior unreported IPV incident with the suspect (80%) and to have ever been strangled (76%) or forced to have sex with the suspect (26%). Effective prevention/detection/intervention strategies are likely to require multidisciplinary collaboration and safety plans that address the susbstantial risk of harm/injury for all adults, children, and animals residing in the home.

Improving prevention of family violence during (and after) disaster: Lessons learned from the Covid-19 pandemic.

As feared by many, our dedicated actions to slow the spread of COVID-19 significantly impacted reporting for most forms of family violence. This concerning decline in reports was greatest during periods of lockdown, when risk of abuse was likely at its highest. A new theory termed, Opportunity to Abuse Theory, helps explain why family violence increased during the Covid-19 pandemic and after most prior natural disasters. The theory focuses on reducing opportunity to abuse by reducing victim vulnerability and increasing perpetrator accountability. Additional actions to improve detection of and reduce risk for family violence, such as improving partnerships with animal welfare organizations (animal abuse reporting was not as impacted during lockdown), enlisting the aid of non-traditional family violence report sources, better utilizing hotels to provide safety when shelter space is limited, and ensuring texting options for reporting abuse, must be considered during and after disaster.

Characteristics of Intimate Partner Violence Incidents and the Environments in Which They Occur: Victim Reports to Responding Law Enforcement Officers.

The objectives of this study were to identify intimate partner violence (IPV) incidence rates, to quantify specific risks and characteristics of these incidents and the environments in which they occur, to identify how often children are present for or injured during these incidents, and to identify differences in victim reports of IPV to law enforcement officers at the scene of the incident compared with previously published reports of IPV from retropsective, anonymous surveys and domestic violence shelter interviews. Data gathered by responding law enforcement officers at the scene of the IPV incident were used to determine the prevalence of IPV incident characteristics and outcomes. Females aged 20 to 39 years, unmarried adults, and African Americans were disproportionately represented as victims of IPV in this study. IPV incidents were significantly more likely to occur on Saturdays and Sundays and during the months of May through August. Relationship durations for suspect-victim pairs were most often less than 12 months at the time of the incident. Weapon use and/or strangulation was common, occurring in 44% of all incidents. Minors (under age 18 years) were frequently present in the home during the IPV incident or a member of the household (59%). This study provides a unique perspective of IPV by utilizing data collected directly from the scene of the incident by first responders. Previously published characteristics of IPV were confirmed, but this study also brings to light new and critical information concerning this prevalent form of violence. Study findings relating to incidence, seasonality, severity, disproportionately affected populations, and child exposure are discussed.

An increasing risk of family violence during the Covid-19 pandemic: Strengthening community collaborations to save lives.

Though necessary to slow the spread of the novel Coronavirus (Covid-19), actions such as social-distancing, sheltering in-place, restricted travel, and closures of key community foundations are likely to dramatically increase the risk for family violence around the globe. In fact many countries are already indicating a dramatic increase in reported cases of domestic violence. While no clear precedent for the current crisis exists in academic literature, exploring the impact of natural disasters on family violence reports may provide important insight for family violence victim-serving professionals. Improving collaborations between human welfare and animal welfare agencies, expanding community partnerships, and informing the public of the great importance of reporting any concerns of abuse are all critical at this time.

Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

More than words: the emotional maltreatment of children.

Emotional maltreatment may be the most complex, prevalent, and damaging form of child maltreatment and can occur simultaneously with other forms of abuse. Children in the first few years of life seem to be at the greatest risk of suffering the most negative outcomes. Medical professionals can help identify and protect victims of emotional maltreatment by carefully observing caregiver-child interactions, paying attention to a family's social history, making referrals to community or counseling programs when necessary, and reporting any suspicions of maltreatment to Child Protective Services. A well-coordinated, multidisciplinary response must be enacted whenever emotional maltreatment is suspected or reported.

Serum-free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential.

There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft-versus-host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC-based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In the present study, we developed and optimized serum-free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum-free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum-containing adherent MSC cultures. Our results suggest that serum-free media for MSC spheroids may pave the way for scale-up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors.

Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions.

The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0±0.2)×105 cells·mL⁻¹ (18±1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4±0.5)×105 cells·mL⁻¹ (14±7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practices-compliant large-scale production system of MSC for cellular therapy.

Does biventricular pacing improve hemodynamics in children undergoing routine congenital heart surgery?

Biventricular (BiV) pacing or cardiac resynchronization therapy (CRT) is an established therapy for heart failure in adults. In children, cardiac dyssynchrony occurs most commonly following repair of congenital heart disease (CHD) where multisite pacing has been shown to improve both hemodynamics and ventricular function. Determining which patient types would specifically benefit has not yet been established. A prospective, repeated measures design was undertaken to evaluate BiV pacing in a cohort of children undergoing biventricular repair for correction of their CHD. Hemodynamics, arterial blood gas, electrocardiographic (ECG), and echocardiographic data were collected. Pacing protocol was undertaken prior to the patient's extubation with 20 min of conventional right ventricular (RV) or BiV pacing, preceded and followed by 10 min of recovery time. Multivariate statistics were used to analyze the data with p values <0.05 considered significant. Twenty-five (14 female) patients underwent surgery at a median (range) age of 5.2 (0.1-37.4) months with no early mortality. The Risk-adjusted classification for Congenital Heart Surgery (RACHS) scores were 2 in 14 patients, 3 in eight patients, and 4 in three patients. None had pre-existing arrhythmias, dyssynchrony, or required pacing pre-operatively. No patient required implantation of a permanent pacemaker post-operatively. The median cardio-pulmonary bypass time was 96 (55-236) min. RV and BiV pacing did not improve cardiac index from baseline (3.23 vs. 3.42 vs. 3.39 L/min/m2; p > 0.05). The QRS duration was not changed with pacing (100 vs. 80 vs. 80 ms; p > 0.05). On echocardiography, the time-to-peak velocity difference between the septal and posterior walls (synchrony) during pacing was similar to baseline and was also not statistically significant. BiV pacing did not improve cardiac output when compared to intrinsic sinus rhythm or RV pacing in this cohort of patients. Our study has shown that BiV pacing is not indicated in children who have undergone routine BiV congenital heart surgery. Further prospective studies are needed to assess the role of multisite pacing in children with ventricular dyssynchrony such as those with single ventricles, those undergoing reoperation or those with high RACHS scores.

Mitochondrial membrane cholesterol, the voltage dependent anion channel (VDAC), and the Warburg effect.

Normal cells of aerobic organisms synthesize the energy they require in the form of ATP via the process of oxidative phosphorylation. This complex system resides in the mitochondria of cells and utilizes oxygen to produce a majority of cellular ATP. However, in most tumors, especially those with elevated cholesterogenesis, there is an increased reliance on glycolysis for energy, even in conditions where oxygen is available. This aerobic glycolysis (the Warburg effect) has far reaching ramifications on the tumor itself and the cells that surround it. In this brief review, we will discuss how abnormally high membrane cholesterol levels can result in a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH-7777). We have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution and transport. Work in our laboratory demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.

The voltage dependent anion channel affects mitochondrial cholesterol distribution and function.

We have observed abnormally high membrane cholesterol levels and a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH7777). Using cholesterol affinity chromatography and MALDI-TOF Mass Spectrometry, we have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution. VDAC is known to associate strongly with hexokinase, particularly in glycolytic cancers. By constructing an E72Q mutant form of VDAC that inhibits its binding of hexokinase, we report an increase in oxidative phosphorylation activity of MH7777 cells, as well as reduced membrane cholesterol ratios to levels near that of normal liver mitochondria. This paper demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.

A cholesterol-binding and transporting protein from rat liver mitochondria.

In this communication, we present results indicating a protein isolated from rat liver mitochondrial intermembrane space that is capable of binding cholesterol and transporting it between the inner and outer mitochondrial membranes. This protein has a molecular weight of 57.5 kDa by SDS-PAGE; however, under native conditions, there is cholesterol-binding capability only as a 115 kDa dimer. Our data show that this dimeric protein may play a role in the regulation of mitochondrial membrane cholesterol levels, a prerequisite for the optimal activity of inner mitochondrial membrane-associated enzyme complexes. In addition, it appears that this protein is largely responsible for the differences in membrane cholesterol levels observed in normal and hepatoma mitochondria, a discrepancy which may help to explain the lack of energy production via oxidative phosphorylation in malignant tumor mitochondria.