Animal Care Committee Research Articles

Training ultrasound-guided percutaneous nephrostomy technique with porcine model.

There is increasing interest in the use of ultrasound for endoscopic and percutaneous procedures. Access can be achieved without radiation exposure under ultrasound guidance. Our aim was to develop a porcine-based training model for ultrasound-guided percutaneous renal access that could also be personalized to a specific patient. The Institutional Animal Care and Use Committee of Severance Hospital approved the study protocol. An anesthetized pig was placed in the dorsal lithotomy position. For the nephrostomy puncture, a Chiba biopsy needle with an echo tip was used under ultrasound guidance. Eight residents and three consultants in urology participated. Puncture time was defined as the nephrostomy time to confirm the flow of irrigation via the needle. After training, satisfaction survey results for clinical usability and procedural difficulty were evaluated. The 5-point Likert scale satisfaction survey for clinical usability and procedural difficulty found mean results of 4.64 and 4.09 points, respectively. There were no differences between residents and consultants for either variable. For all participants combined, there was a significant difference for nephrostomy time between the first and second trials (278.8±70.6 s vs. 244.5±47.0 s; p=0.007). The between-trial difference was greater for residents (291.5±71.2 s vs. 259.1±41.9 s; p=0.039). The difference for the consultant was not significant (245.0±69.4 s vs. 205.7±42.5 s; p=0.250). We developed a porcine-based ultrasound-guided nephrostomy puncture training model. Satisfaction survey results indicated high clinical usability and procedural difficulty. For nephrostomy time, the model was more effective for urology residents than for consultants.

A survey of the impact of COVID-19 on the management of animal experiments and laboratory animal facilities in Korea.

The pandemic has affected the lives of people all over the world. The effects of the pandemic on laboratory animal facilities and their operations through this unusual global event are poorly understood. Here, we have applied a methodological framework of qualitative approach including semi-structured interviews to investigate laboratory animal operations in Korea and how it has shaped the on-going management and laboratory operations of such facilities. A total of fifty-two individuals, including members and administrators of the Institutional Animal Care and Use Committee (IACUC), researchers, and animal facility managers and staff, were surveyed through purposeful sampling. Survey questions explored how the pandemic impacted the IACUC and the functioning of animal facilities, and what steps to take in preparation of a future pandemic-like crisis. Our survey found evidence of an increase in animal experiments in Korea during the pandemic that correlated with increases in research funding during that period, such as for vaccine development. Also, operational challenges due to pandemic-related health issues in personnel were resolved through overtime, rather than by reducing facility operations. Moreover, a refinement of post-approval monitoring (PAM) practices was also discussed by respondents. Taken together, our study offers insights into animal facility operations during the pandemic and outlines recommendations for safeguarding operations in such future scenarios.

A Commentary on Fasting of Nonclinical Research Animals.

This commentary discusses the implementation of fasting in nonclinical animal experimental subjects. The short-term removal of food from cages of experimental animals is in all respects innocuous. The term "stress" is ill-defined and the statutes and regulations governing animal research laboratories that exert their authority in the performance of their operations do so without substantive grounds to base compliance. The legislative and administrative history of the implementation of the Animal Welfare Act (AWA) has evolved into the development of laboratory management strategies that focus on the reduction of the biological cost of stress to the animals and the determination of when subclinical stress (eustress) becomes distress. Animal welfare is based on the tenet that in laboratories conducting animal research in compliance with Good Laboratory Practices (Title 21 USC, Chapter 13,§58), it is the study protocol and the study director that establish procedures and processes that are approved by each Institutional Animal Care and Use Committee to ensure the humane care and use of animals in research, teaching, and testing and to ensure compliance with guidelines and regulations. This approval process establishes the justification of eustress in the environment that do not rise to the threshold of distress under the AWA.

Endogenous Protein C Activation with AB002 Improves Outcomes in a Rat Model of Acute Ischemic Stroke

Background: The hemorrhagic side-effects of current thrombolytic agents limit their usefulness to a small subset of acute ischemic stroke patients. An early, effective, and hemostatically safe antithrombotic/neuroprotectant in addition to standard of care could improve outcomes. AB002 (E-WE thrombin) is an engineered recombinant thrombin analog (W215A/E217A) that activates cytoprotective and antithrombotic endogenous protein C on intravascular surfaces without any measurable procoagulant activity. In renal failure patients undergoing hemodialysis, AB002 treatment reduced dialyzer clotting without increasing markers of hemostasis impairment or coagulation (NCT03963895). Preclinical studies also showed that AB002 exhibits potent antithrombotic/thrombolytic effects in a non-human primate model of acute vascular graft thrombosis, as well as reduces infarct size and improves outcomes in an experimental acute myocardial ischemia-reperfusion model in mice. Aims: Here, we evaluated whether neuroprotective effects of AB002 can be observed following middle cerebral artery occlusion-recanalization (MCAO-R) in rats. Methods: The local animal care and use committee approved all studies. 8 to 9-week-old male rats (275-377 g) were anesthetized under isoflurane and a small laser-Doppler probe was affixed to the skull to monitor cortical perfusion and verify vascular occlusion and reperfusion. A silicone-coated 4-0 nylon monofilament was inserted into the right internal carotid artery (ICA) via the external carotid artery (ECA) until the laser-Doppler signal dropped to < 30 % of baseline. To evaluate the effect of AB002 on outcomes, rats (n = 4/ group) were treated with 25 µg/kg (0.25 mL/kg; i.v.) or saline placebo, administered 30 min after induction of ischemia. Since prolongation of the activated partial plasminogen time (aPTT) is a pharmacologic effect of high dose drug exposure, a 0.45 mL blood sample was collected at 20 min post treatment from the right jugular vein, processed to plasma and frozen for subsequent aPTT evaluation. At 90 min, the filament was removed, the surgical incisions were sutured closed and postoperative animals were monitored during recovery. Neurological performance scores and weight were assessed daily for 4 days. Rats were sacrificed on day 7 post-surgery and infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. To account for the effect of edema, the infarcted area was estimated indirectly by subtracting the non-infarcted area in the ipsilateral hemisphere from the contralateral hemisphere and expressing infarct volume as a percentage of contralateral hemisphere. APTT was measured in duplicate using a KC-4 coagulometer. All analyses were performed by researchers blinded to treatment. Results: Compared to placebo, rats that received AB002 exhibited less ischemic damage, evidenced by a statistically significant reduction in infarct volume of the cortex (32.3% versus 8.9%, P < 0.05) and hemisphere (28.0% versus 9.8%, P < 0.01). In addition, rats had better neurological scores and greater weight gains during recovery following treatment with AB002, compared to placebo. Mean aPTT values were higher in AB002 treated rats compared to placebo (34 ± 2.2 s versus 21 ± 1.6 s), confirming drug exposure. Conclusions: Treatment with AB002 during ischemia reduced infarct volume and improved outcomes in the rat experimental stroke model. Considering its neuroprotective and antithrombotic properties, we propose that early treatment with AB002 may confer outcome benefit for patients experiencing acute thrombotic emergencies.

1954. Dynamic PET facilitated Modeling and Novel Antibiotic Regimens for Tuberculous Meningitis due to Multidrug-Resistant Strains

1954. Dynamic PET facilitated Modeling and Novel Antibiotic Regimens for Tuberculous Meningitis due to Multidrug-Resistant Strains

Knowledge, Attitudes, and Practices on Ethics in Biomedical Animal Research in Mexico.

The most widely accepted ethical concept for the mitigation of harm to animals used in biomedical research is known as the 3Rs, which refer to replacement, reduction, and refinement. The aim of our study was to determine the ethical and regulatory criteria that researchers in Mexico consider when developing their animal research protocols and that members of the ethics committees use when they evaluate and approve these protocols. We circulated a survey to 300 individuals from different research institutions and received responses from 179 researchers and members of ethics committees on questions related to their knowledge, attitudes, and practices toward the use of animals in research based on the 3Rs. The responses obtained indicate that the respondents were aware of the 3R concept, and they claim to apply these principles. However, the responses revealed resistance to using alternatives for research, testing, and teaching (66%). Nineteen percent of the researchers reported that their institutions do not have an integrated Institutional Animal Care and Use Committee (IACUC). Around 80% of respondents were aware of Mexican regulations. The knowledge and application of the 3Rs by researchers and members of the IACUC is a fundamental concept in animal research. Such knowledge contributes the use of ethical standards, attitudes, and practices relevant to the use of animals in research.

Cardiosphere derived cell secretome therapy after experimental myocardial infarction: effects of intracoronary administration on ventricular tachycardia inducibility and cardiac function

Abstract Background Myocardial infarction (MI) carries high risk of sudden cardiac death due to the development of ventricular tachycardia (VT) which is caused by viable myocytes located within the infarct scar tissue [1]. Cardiosphere derived cell secretome (S-CDCs) has been demonstrated to possess antiarrhythmic properties when delivered transendocardially [2]. Further, S-CDCs has been proven to reduce scar size and increase viable myocardial mass in chronic MI [3]. Purpose The objective of this study was to evaluate the effects of an intracoronary (IC) administration of S-CDCs on VT inducibility and cardiac function in a porcine MI model. Methods After obtaining the approval of the Institutional Animal Care and Use Committee, 14 Large White swine were subjected to closed chest MI induction carrying out balloon occlusion of the left anterior descending coronary artery (below the second diagonal branch) for 150 minutes. One month after MI creation, pigs received an IC administration of saline (CON; 5mL; n=7) or S-CDCs (S-CDCs; 9.16 mg of protein in 5mL of saline; n=7). VT inducibility and magnetic resonance (MR) studies were performed pre- and four months post-injection determining ejection fraction (EF), infarct size (IS) and indexed end diastolic and systolic volumes (EDVi, ESVi). Results Before injection VT was inducible in all cases and no differences were observed between groups in any MR-derived parameter (figure). IC infusion was successful in all swine. At four months VT inducibility study revealed lower inducibility rates in S-CDCs compared to CON (57% versus 100%; p=0.05). At this timepoint EF was higher in S-CDCs (35±10% versus 29±10%) although differences between groups were not statistically significant. Conversely, IS was significantly lower in S-CDCs (12±3% versus 16±2% (p=0.03)). Moreover, a trend towards lower ventricular volumes (EDVi: 83±18mL/m² versus 88±29mL/m² and ESVi: 56±20mL/m² versus 64±27mL/m²) was detected in this group. Conclusions The IC administration of S-CDCs suggests a potential reduction on post-MI VT development as well as a beneficial effect on cardiac function. This therapy seems to reduce IS in this porcine MI model.MR derived parameters

Abstract 201: 3D-Printed Automated Pressure-Cycled Resuscitator Maintains Respiratory Homeostasis in Swine With Normal to Moderately Low Lung Compliance

Background: During resuscitation, manual ventilation is commonly used. However, the effectiveness of this approach heavily relies on the skills of the individuals performing, often leading to inconsistent tidal volumes and respiratory rates. Such variations in ventilation can disrupt the removal of CO 2 resulting in altered pH. To address this problem, we leveraged advancements in fluidics and 3D printing to develop a miniature, automated, pressure-cycled resuscitator (inVent) that operates without any moving parts or settings. Hypothesis: The inVent will be able to maintain a pH of 7.2 or higher in swine with a dynamic compliance (Cdyn) of 25-50 ml/cmH 2 O. Goals/Aims: To assess the inVent’s performance in an animal model with normal to severe lung injury. Methods: Hybrid Landrace swine were studied under a protocol approved by the Institutional Animal Care and Use Committee. All swine were anesthetized, and underwent oral intubation. Breath by breath measurements were obtained using the NM3 Monitor (Phillips). All 3 inVent (fluidIQ) resuscitators pressure modules were used pre and post lung injury to fully characterize how each of the modules would react in different lung conditions. Lung injury was induced through a repeated bronchoalveolar lavage. ABG analysis was performed using the epoc system (Siemens). pH values 7.2 or less were considered acidotic while those over 7.2 were considered acceptable. Results: 8 swine with an average weight of 36 (5.8) Kg were studied. 7 swine contributed to 39,770 breaths of pulmonary mechanics and 33 ABGs recorded. Mean Cdyn was lower than anticipated at 25.8 (14.7) mL/cmH 2 O and a pH 7.32 (0.16). Figure 1 demonstrates a linear relationship with compliance and pH. Conclusions: The inVent resuscitator was able to maintain a pH of at least 7.2 in moderate compliance ranges, however struggled to maintain pH of 7.2 or higher in the severely injured. Higher lung compliance ranges are needed to explore possible hyperventilation events.

Abstract 11774: Right Ventricular Function and Myocardial Blood Volume Analyses by Convolutional Neural Network in Experimental Pulmonary Hypertension

Introduction: Survival of patients with pulmonary hypertension (PH) is highly dependent on the right ventricular (RV) function. Despite the association of various factors with adaptive or maladaptive RV phenotypes, the contribution of microvascular density to RV adaptation to pressure overload remains unclear. Hypothesis: This study aimed to evaluate the RV myocardial blood volume and its role in the RV adaptation to pressure overload. Methods: Both the University Animal Care Committee and the federal authorities for animal research of the Regierungsprasidium Giessen (Hessen, Germany) approved the study protocol. The study used adult Wistar-Kyoto rats and Sprague-Dawley rats to induce PH using SuHx and monocrotaline (MCT), respectively. Echocardiography, in vivo contrast-μCT, and invasive hemodynamic measurements were performed. The researchers used an artificial intelligence (AI)-based convolutional neural network (CNN) approach to reconstruct, segment, and analyze the cardiac μCT images. The RV functional reserve was evaluated during mCT acquisition and dobutamine stress test, and metabolomics analysis was performed. Results: The data showed that SuHx rats developed maladaptive cardiac function and RV-PA uncoupling. MCT rats exhibited adaptive RV function at days 14 and 21, but maladaptive function at day 35, which was associated with RV-PA uncoupling and reduced RV functional reserve. Impaired PKA-CREB signaling pathway was also found to be associated with the reduced RV functional reserve in the MCT rats. In both PH models, the absolute RV blood volume significantly increased with the development of RV hypertrophy, but the relative blood volume decreased, which was associated with maladaptive RV function Conclusions: This study is the first to demonstrate the use of AI CNN in post-processing of μCT-derived cardiac images from experimental PH models. Additionally, a single-beat PV loop was established for the first time in a rat model of PH by combining non-invasive and invasive techniques. The findings suggest that RV maladaptive function in rat models of PH is characterized by capillary rarefaction, impaired RV reserve in response to dobutamine infusion, which is associated with impaired PKA-CREB signaling pathway in the RV.

PSVIII-22 A Probiotic Blend Improves Fecal Quality and Enhances Gut Immunity in Dogs with Chronic Diarrhea

Abstract Probiotics have been fed to pets for their enhanced health benefits beyond basic nutrition, as well as to manage diarrhea. Species and strains of probiotics and the combinations may elicit different beneficial effects in dogs. Therefore, the objective of this study was to evaluate the effects of a probiotic blend (2 strains of Bacillus subtilis and 1 strain of Bacillus amyloliquefaciens) on digestibility, fecal characteristics, fecal microbiota, and fecal metabolites in healthy dogs and dogs with chronic diarrhea (CD = dogs with intermittent diarrhea persisting for more than 3 months before study start that were otherwise clinically healthy). Twenty-two adult dogs (11 healthy, 11 CD; mean BW = 14.1 kg; mean age = 8.1 yr) were enrolled in a cross-over design study, with two 3-week feeding periods and a 3-week washout between feeding periods. Protocols were approved by the facility’s Institutional Animal Care and Use Committee. Dogs were assigned to a control kibble or control kibble + probiotic blend (1.5 × 106 CFU/g) top dressed on the food. Fecal scores were evaluated daily during each period. Fecal samples were collected during days 14 to 18 for nutrient digestibility. Fresh fecal samples were collected on days 17 to 18 for dysbiosis index and fecal metabolites measurements. All data were analyzed using the Mixed Models procedure of SAS. Probiotic supplementation did not affect digestibility, dysbiosis index, or fecal volatile fatty acid concentrations in both healthy and CD dogs. Fecal scores were lower (P < 0.05; firmer stool) when dogs consumed the probiotic blends for both healthy and CD groups. In dogs with CD, consumption of the probiotic blend resulted in greater (P < 0.05) fecal IgA concentrations. These data demonstrate that the probiotic blend may elicit intestinal health benefits by improving stool quality and enhancing gut immunity, especially in dogs with CD.

200 The Effects of Whole Hemp Seed Supplementation on Health Status, Voluntary Physical Activity, Fecal Quality, and Fermentative End-Products in Healthy Adult Dogs

Abstract The cultivation and utilization of hemp and hemp-derived products have increased at a rapid pace since the enactment of the 2018 farm bill which provided changes in regulation related to cannabis. Although there are three hemp-derived ingredients that have been generally recognized as safe status for human consumption by the FDA, no ingredients have been approved by the FDA for consumption by companion animals or defined by AAFCO. Hemp seeds have the potential to act as a rich source of plant protein, polyunsaturated fatty-acids, and dietary fiber in companion animal nutrition. The objective of this study was to evaluate the effects of supplementing ground whole hemp seed in a baked chew on voluntary physical activity, fecal characteristics, fermentative-end products, and overall health status in healthy adult dogs. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee and were in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Thirty adult beagle dogs were used in a complete randomized design, with ten dogs assigned to each treatment. The dogs were fed the same commercially available, complete and balanced diet and were supplemented with one of three baked chews containing either a high inclusion of ground hemp seed (50%), a low inclusion of ground hemp seed (25%), or cellulose. The baked chews were fed at a level of 10% of the daily intake for 28 d and were formulated to be similar in macronutrient composition, approximately 25% fat, 20% protein, and 25% total dietary fiber. Duration and intensity of voluntary physical activity was measured using activity monitors for 4 d before supplementation and at the end of the supplementation period (d 24 to 27). Fasted blood and a fresh fecal sample were taken on d 0, 14, and 28 to monitor health status, blood variables, fecal characteristics and fermentative-end product concentrations. The baked chews were well accepted by the dogs with minimal intake refusals, and all dogs remained clinically healthy for the duration of the experimental period. No differences in fecal score or fecal pH were observed among the treatment groups (P > 0.05). The treatment did not have an effect on fecal concentrations of volatile fatty acids including acetate, propionate, butyrate, valerate, isovalerate, and isobutyrate (P > 0.05). Concentrations of fecal ammonia, phenols, and indoles were also similar among the groups (P > 0.05). No adverse effects were observed, and the data suggest that in a baked chew the whole ground hemp seed was comparable to the cellulose control.

PSXIII-27 Evaluation of Protein Quality of Select Chicken-Based Protein Sources Using the Precision-Fed Cecectomized Rooster Assay

Abstract The objective of this experiment was to determine and compare the standardized amino acid (AA) digestibility of chicken-based protein sources using the precision-fed cecectomized rooster assay. Four ingredients were tested: spray-dried chicken protein hydrolysate (CPH), spray-dried chicken meat (CC), spray-dried chicken broth (CB), and chicken meal (CM). All animal care procedures were approved by the University of Illinois Institutional Animal Care and Use Committee before experimentation. Sixteen cecectomized roosters (4 roosters per substrate) were randomly assigned to the four test substrates. After 24 h of feed withdrawal, roosters were tube-fed 12 g of test substrates and 12 g of corn. Following crop intubation, excreta (urine and feces) were collected for 48 h. Endogenous corrections for AA were made using 5 additional cecectomized roosters. All data were analyzed using the Mixed Models procedure of SAS version 9.4. Generally, CC had the greatest (P < 0.05) standardized digestibility for AA, followed by CPH. Methionine, phenylalanine, and valine had greater (P < 0.05) digestibility in CC and CPH compared with CB and CM. Digestibility of isoleucine and leucine were greater (P < 0.05) in roosters fed CC, followed by CPH, CM, and CB. Greater digestibility (P < 0.05) of lysine was observed in roosters fed CC and CPH, followed by CM and CB. Histidine and threonine had the least digestibility (P < 0.05) in CB compared with all other ingredients. In contrast, digestibility of tryptophan was greatest (P < 0.05) in CC, followed by CPH and CB, while CM had similar (P > 0.05) digestibility to CC and CPH. Amino acid digestibility for most indispensable AA was above 80% for all ingredients, except for histidine, lysine, and threonine, which were exceptions only for CB (68%, 73%, and 74%, respectively). For selected dispensable AA, amino acid digestibility was above 60% for all ingredients, except for aspartic acid in CB and CM, and serine in CB, which were exceptions (43, 68% and 66%, respectively). In general, the least DIAAS values were observed in roosters fed CB, followed by CPH, CM, and greatest in CC. Overall, CC and CPH have comparable indispensable AA digestibility, and tryptophan or phenyl alanine+tyrosine were, generally, the first limiting AA.

PSVIII-20 A Premium Dry Cat Food Enhances Digestibility and Gut Health in Cats Compared with a Grocery Dry Cat Food

Abstract As the awareness of pet owners to the benefits of nutrition on pet health and wellness increases, pet owners are looking for foods that include high-quality ingredients and functional benefits. However, little research is available on cats comparing health measures when feeding premium foods vs basic cat foods. This study aimed to compare the effects of feeding a premium cat kibble diet (Blue Buffalo Tastefuls; BB) and a basic grocery cat kibble diet on digestibility, blood biomarkers, and fecal microbiome in healthy adult cats. Sixty cats (mean BW = 4.2 ± 0.8 kg; mean age = 4.3 ± 3.2 yr) were randomly selected to consume BB (n = 30) or grocery diet (n = 30) for 180 days. On days 0, 90, and 180, fresh fecal samples were collected for fecal microbiome and dysbiosis index analysis and blood samples were collected for measurements of complete blood count, serum chemistry, and serum biomarkers. A separate 10-day digestibility study was conducted using 14 cats (BB n = 7, grocery diet n = 7; mean BW = 3.8 ± 0.7 kg). Total fecal samples were collected from day 6 to day 10 for nutrient digestibility analysis. Protocols were approved by the facility’s Institutional Animal Care and Use Committee before the study. All data were analyzed using the Mixed Models procedure of SAS. Fecal scores were not changed (P > 0.05) by diet. Greater (P < 0.05) digestibility of protein, fat, and energy were observed in cats consuming BB. Although all blood cell count and blood chemistry values were within the reference ranges, measurements such as total protein, aspartate transaminase, alkaline phosphatase, cholesterol, and triglycerides were significantly different (P < 0.05) between diet groups. Greater (P < 0.05) insulin-like growth factor-1 was observed in cats fed BB. Cats consuming BB had a lower (P < 0.05) dysbiosis index (less dysbiotic microbiome) and a greater (P < 0.05) bacterial species richness. Relative abundances of 28 bacterial genera were altered (P < 0.05) by the diets, with greater abundances of Faecalibacterium, Megamonas, and Ruminococcus observed in cats fed BB. The data suggest that a premium dry cat food may elicit beneficial effects on digestibility and the GI microbiome of healthy adult cats.

PSI-9 Efficacy of Electrolyte Chews in Horses

Abstract Oral supplementation with electrolytes is commonly practiced among horse owners. However, practical ease of administration can be challenging due to palatability issues. The objective of this study was to investigate the efficacy of a novel electrolyte product form during routine exercise. Protocol approval (21-029) by Southern Illinois University Institutional Animal Care and Use Committee was obtained before initiation of this work. Privately-owned local foxhunting horses (n = 15) were evaluated. Treatment groups were identified based on voluntary consumption of chews and include control (CON) = 0 chews; light (LIG) 6 to 9 chews; and moderate (MOD) 15 to 20 chews. Jugular venipuncture and vitals assessments were completed 30 minutes before and 30 minutes following a 2-hour ride. Electrolyte chews were offered in two sessions: 30 minutes pre-ride and immediately post-ride. Serology values for Na, K, Cl, Ca, HCO−3, Mg were measured as well as respiratory rate (RR), skin turgor (SKN), and rectal temperature (BT). Categorical variables included body condition score (BCS), sex, and breed (BRD). Data were analyzed via PROC GLM, PROC FREQ and PROC CORR dependent on test with SAS (v. 9.5). Significance for all tests was set at P < 0.05. Serological values were unaffected by electrolyte consumption (P > 0.05). Body temperature was significantly higher for all horses following exercise, regardless of treatment group (P = 0.0005). Skin turgor showed significantly more time before return to original state during post-ride evaluation (P = 0.0013). Respiratory rate was noted to decrease post-exercise, suggesting an increased baseline due to recent transport (P = 0.0158). Directional serum changes between Cl and K were correlated (P = 0.0227) as well as Mg and Cl (P = 0.0316). Correlations of relational change between Ca and HCO−3 in addition to Ca and Na were approaching significance (P = 0.0707; P = 0.0800). Mares demonstrated a greater ability to maintain lower post-ride serum calcium levels than geldings (P = 0.013). Preliminary effect of breed on serological calcium change may also be considered (P = 0.0312). To conclude, differences in sex may alter electrolyte use pathways, such as calcium. The exercise challenge was sufficient based on vitals assessments. These data suggest key factors associated with chewable electrolyte form administration. Further research is needed to identify the impacts of electrolyte form on efficacy as well as improve palatability.

PSX-10 Pilot Study Estimating Energy Expenditure in Dogs Using a Quantified Health Approach Indicates Potential to Improve Estimation of Energy Requirements

Abstract Over 50% of dogs in the USA are overweight or obese indicating that owners find it difficult to know how much food their dog needs. Feeding guidelines are based on the average energy requirements of dogs and do not take into account measured activity levels, which may lead to overfeeding by well-intentioned owners. The most common published energy prediction equation is (RER*AF) where RER is Resting Energy Requirement [70*(BW kg)0.75)] and AF is the Activity Factor, which generally ranges between 1 (body weight loss) and 8 (heavy work). This equation requires the user to estimate activity levels when selecting an AF, which may result in under- or overfeeding. The objective of this pilot study was to determine the ability of collar-worn activity monitors to more accurately predict the energy expenditure of dogs. Ten weight-stable beagles were group-housed at the Hill’s Pet Nutrition Center in Topeka, KS. Dogs were provided daily access to open, outdoor play space and human interaction; water was freely available. Dogs were weighed before feeding on days 1 and 5 of each of the 2 test periods. Accelerometer-based activity monitors were attached to the collars of the dogs on day 1 of each test period and activity was continually measured throughout the study using the activity monitor. Daily food consumption in grams was recorded and was converted to energy intake using the caloric density of the food. Daily energy requirements were calculated for each dog using the standard RER equation and either a standard AF of 1.6 or the estimated energy required for activity, in which newly established algorithms for quantification of walking, trotting, and running behaviors were paired with estimated caloric expenditure based on level of exertion (quantified health approach). The mean absolute error (MAE) of each method was calculated as the absolute value of the difference between the daily energy requirement vs. the daily caloric intake for each dog. The MAE was averaged across all dogs and all days for each of the two methods. The MAE ± SD of the standard RER and AF was 103 ± 126 kcal/day (Range -21.5 to 216 kcal/day). The MAE ± SD of the quantified health approach was 86 ± 90 kcal/day (Range -37.6 to 164.4 kcal/day). Both approaches resulted in normal MAE distributions. In this pilot study, quantifying energy expenditure using a wearable activity monitor reduced over-feeding by 16.5% compared with standard equations. In the future, energy predictions based on measured activity could result in more accurate feeding recommendations than traditional approaches. Future research aimed at optimizing energy predictions may help address the problem of pet obesity. This study was approved by the Hill’s Internal Animal Care and Use Committee. Funding: This study was funded by Hill’s Pet Nutrition, Inc.

PSVIII-21 A Premium Dry Dog Food Elicits Gut Health Benefits Compared with a Grocery Dry Dog Food

Abstract With the pet humanization trend, pet parents are continuously seeking approaches to improve the health of their pets. Compared with grocery dog foods produced to meet the basic needs of the dog, premium dog foods include high-quality ingredients and functional benefits that can be more appealing to consumers. However, little research is available comparing health measures in dogs fed premium foods vs. basic grocery foods. The objective of this study was to compare the effects of feeding a premium dog kibble diet (Blue Buffalo; BB) and a grocery dog kibble diet on digestibility, blood biomarkers, and fecal microbiome in healthy adult dogs. Sixty dogs (mean BW = 10.8 ± 2.0 kg; mean age = 8.2 ± 3.3 yr) were randomly selected to consume BB (n = 30) or the grocery diet (n = 30) for 180 days. On days 0, 90, and 180, fresh fecal samples were collected for fecal microbiome and dysbiosis index analysis and blood samples were collected for measurements of complete blood count, serum chemistry, and serum biomarkers. A separate 10-day digestibility study was conducted using 12 dogs (BB n = 6, grocery diet n = 6; mean BW = 12.0 ± 2.1 kg). Total fecal samples were collected from day 6 to day 10 for nutrient digestibility analysis. Protocols were approved by the facility’s Institutional Animal Care and Use Committee before the study. All data were analyzed using the Mixed Models procedure of SAS. Stool quality was not different (P > 0.05) between diet groups. Both diets were highly digestible without dietary effects (P > 0.05). All blood cell count and serum chemistry values were within the reference ranges. However, dogs fed the grocery diet had greater (P < 0.05) liver enzyme concentrations including alanine transaminase, alkaline phosphatase, and gamma-glutamyl transpeptidase, and greater (P < 0.05) monocyte counts compared with BB. Dogs had greater (P < 0.05) serum pancreatic polypeptide when fed BB. A lower dysbiosis index (less dysbiotic microbiome) was observed in dogs consuming BB. Relative abundances of 23 bacterial genera were altered (P < 0.05) by the diets, with greater abundances of Bifidobacterium and Megamonas and decreased abundance of Streptococcus observed in the BB group compared with the grocery diet group. These results suggest that premium dry dog food could potentially elicit a positive impact on blood parameters and GI health.

186 Amino Acid Digestibility of Various Protein-Based Ingredients Using the Precision-Fed Cecectomized Rooster Assay

Abstract Given their high protein quality and sustainable production practices, there is growing interest in using single cell-based proteins in pet foods. Because there has been a lack of testing on these proteins, the objective of this experiment was to determine the amino acid (AA) digestibilities of a single cell protein and other protein-based ingredients using the precision-fed cecectomized rooster assay. Test ingredients included: Calysta microbial protein (MP), chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae (BSFL). All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation. Thirty cecectomized roosters (n = 6/ingredient) were randomly assigned to test ingredients. After 24 h of feed withdrawal, roosters were tube-fed 15 g of test ingredient and 15 g of corn. Following crop intubation, excreta were collected for 48 h. Endogenous corrections for AA were made using 5 additional cecectomized roosters. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to AAFCO nutrient profiles, FEDIAF nutritional guidelines, and NRC recommended allowances for adult dogs, adult cats, growing puppies, and growing kittens. Data were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant. All of the reactive lysine:total lysine ratios, an indicator of heat damage, were greater than 0.9 except for CM (0.86). All indispensable AA had digestibilities >85% for MP, with indispensable AA digestibilities being >80% for all ingredients. In general, CGM had the greatest AA digestibilities, while CM had the least AA digestibilities. Two exceptions were the digestibilities of lysine and tryptophan, which were greatest in MP. Lysine digestibility for MP (92.3%) was greater (P < 0.01) than that of all other ingredients. Tryptophan digestibility for MP (97.2%) was greater (P < 0.0001) than that of CM, CGM, and PP. Threonine and valine digestibilities were also greater for MP. Threonine digestibility was greatest for CGM (91.0%) and MP (88.6%), being greater (P < 0.0001) than that of the other ingredients. Valine digestibility was greatest for CGM (93.3%), PP (89.3%), and MP (89.3%), being greater (P < 0.001) than that of CM. DIAAS-like calculations estimated the protein quality of each ingredient, with the limiting AA of each depending on the reference used (NRC; AAFCO; FEDIAF) and life stage (growing; adult) and species (dog; cat) of animal in question. For dogs, limiting AA were most commonly methionine, threonine, and tryptophan. For cats, limiting AA were most commonly lysine and methionine. Further research in dogs and cats is necessary to confirm sufficient palatability and digestibility. However, our data suggest that the MP tested is a high-quality protein source with high AA digestibilities, and may serve as a valuable protein alternative for pet foods.

PSVIII-19 Saccharomyces Cerevisiae Fermentation Product Modulates Blood Cell Counts and Gut Immunity in Healthy Adult Cats

Abstract Dietary Saccharomyces cerevisiae fermentation product (SCFP) has been shown to modulate immune responses in dogs and livestock. Information about feeding SCFP to cats, however, is lacking. This study aimed to evaluate the effects of SCFP (TruMune, Cargill, Inc.) on immune indices including fecal IgA, serum oxidative stress markers, and toll-like receptor (TLR) responsiveness in healthy adult cats. Sixty-three cats (mean age = 6.3 ± 2.8 yr; mean BW = 4.4 ± 0.1 kg) were used in a randomized complete block design. All cats were fed a control kibble diet without SCFP (CON) during a 21-day adaptation period. Cats were randomly assigned to one of the three diets including CON, a dry diet containing 150 mg/kg BW SCFP (TRUL), and a dry diet containing 300 mg/kg BW SCFP (TRUH) and fed for 42 days. Fecal samples were collected on days 0, 21, and 42 for fecal IgA analysis. Serum samples were collected on days 0 and 42 for measurements of serum chemistry, complete blood count, and oxidative stress markers. Whole blood samples were collected on day 42 for TLR responsive evaluation. Protocols were approved by the facility’s Institutional Animal Care and Use committee before the study. Data were analyzed using R v.4.1.2. Differences P < 0.05 were considered significant and P < 0.10 as trends. All serum chemistry and blood cell count values were within the reference ranges, with serum chemistry not affected by SCFP. Absolute cell counts of eosinophils and neutrophils decreased linearly with SCFP dose (P < 0.001) while absolute cell counts of lymphocytes and monocytes increased linearly with SCFP dose (P < 0.01). TLR responsiveness was not altered (P > 0.05) by SCFP. A tendency (P = 0.08) of quadratic treatment dose response was noted for thiobarbituric acids from the thiobarbituric acid reactive substance assay, with cats fed TRUL having the greatest thiobarbituric acid values. Fecal IgA showed a quadratic treatment dose response on day 42 (P = 0.01), with cats fed TRUL having the lowest fecal IgA values. These results demonstrate that SCFP may affect immune outcomes including blood cell counts and fecal IgA in adult healthy cats.

Mildronate Ameliorates Kidney Function and Reduces Inflammation in SCD Mice

INTRODUCTION:Sickle cell disease (SCD) is a genetic hemoglobin disorder characterized by hemolytic anemia, vaso-occlusive crisis and inflammation that affects organs such as the kidney, liver, brain, spleen, and heart. SCD is driven by a mutation in the β-globin gene that causes sickling of red blood cells (RBCs). More than 50% of SCD patients are affected by chronic kidney diseases (CKD) and other comorbidities. Vasculopathy is caused by hemolysis-related endothelial dysfunction and inflammation, which collectively contribute to the development of renal disease. Mildronate (also known as Meldonium) is an anti-ischemic and cardio protecting medication approved in Eastern Europe. Mildronate facilitates a switch from fatty acid oxidation to less oxygen-intensive glycolysis that allows less oxygen consumption by peripheral tissues and higher oxygenation of heart. We hypothesized that mildronate administration would reduce tissue hypoxia and decrease inflammation in SCD mice. METHODS: The study was approved by the Institutional Animal Care and Use Committee at Howard University (IACUC). We employed the Townes SCD mouse model, which mimics SCD complications. SCD and control mice were injected with mildronate (400 mg/kg in saline) intraperitoneally or saline as vehicle control over the course of ten days. Blood and plasma were collected and used for hematological parameters analysis. Cytokines were measured by Bio-plex suspension array in plasma and real-time RT-PCR or ELISA in organs. Protein expression of HO-1, HIF-1α, NFκB and NRF2 was measured by Western blot. The effect of Mildronateon systemic and renal oxidative stress was measured by malondialdehyde (MDA) using ELISA (Abcam). RESULTS: We observed significant reduction in the cytokine's levels of IFN-γ, IL-1, and TNF-α in plasma of SCD mice after mildronate injection. We also observed reduction in IL-6, IL-10 and IL-17 levels but differences were not statistically significant. There were no significant changes in hematological parameters (Hematocrit, MCV, MCH, CMCH, Erythrocytes, Hemoglobin) after mildronate injection. Also, we did not observe changes in MDA levels in plasma or kidney in SCD mice compared to control mice, both non-injected and mildronate injected. We further evaluated HO-1 and NRF2 protein expression by western blot and observed no significant differences between SCD mice with or without mildronate injection. In H&E staining, cortex glomerular area and medullar congestion area were significantly reduced after mildronate injection. We also observed reduction of proteinuria in SCD mice injected with mildronate. DISCUSSION: Our examination of the effect of mildronate showed decreases inflammation in the mildronate injected SCD mice evidenced by reduced levels of IFN-γ, IL-1, IL-17, IL-6, IL-10, and TNF-α in circulation. There was a slight decrease in the expression levels of Nrf2 and HO-1 after mildronate injection. HO-1 is crucial for the catabolism of hemoglobin and protects against tissue damage in hemolysis. The HO-1 promoter has binding sites for several transcription factors, including phosphorylated Nrf2. Previous studies from our lab showed increased HIF-1α expression in PBMCs obtained from SCD patients. Here we did not detect significant changes in NFκB-p65 or HIF-1α expression levels. While we expected mildronate to decrease the oxidative stress, we did not observe significant reduction in MDA levels in treated mice compared to control mice. CONCLUSION: Mildronate improved kidney function in SCD mice by reducing circulatory and renal inflammation. In future, we plan to assess in more details the mildronate's impact on kidney function and inflammation. ACKNOWLEDGMENTS: This work was supported by NIH grants 1R01HL125005, 5U54MD007597, and 1SC1HL150685.

Protocol for the PATHOME study: a cohort study on urban societal development and the ecology of enteric disease transmission among infants, domestic animals and the environment

IntroductionGlobal morbidity from enteric infections and diarrhoea remains high in children in low-income and middle-income countries, despite significant investment over recent decades in health systems and water and sanitation infrastructure....