Rafael Frias

Assessment of practical skills is a key component in ensuring competence in laboratory animal science (LAS), particularly for practitioners performing functions A, C and D as defined in the Directive 2010/63/EU, namely those who carry out procedures on animals, care for them and perform humane killing, respectively. To address the need for a harmonised, objective and transparent approach to assessing practical competence, Working Group 4 (WG4) of the Education and Training Platform for Laboratory Animal Science (ETPLAS) has developed a set of Direct Observation of Procedural Skills (DOPS) tools. These tools are tailored specifically to evaluate the intended learning outcomes of Modules 3.2, 6.2, 8 and 20–22 of the European Union (EU) Education and Training Framework. This report outlines the rationale, structure and guidance for implementing the DOPS tools. Based on Miller’s pyramid and established educational principles, DOPS tools are suitable for formative and summative assessments in simulated or real environments. They can be used both formatively to monitor progress during training and summatively to support end-of-training decisions about competence. Their implementation is crucial to supporting harmonised training, facilitating mutual recognition of competence and ultimately advancing both animal welfare and the quality of scientific research in the EU and beyond. The DOPS forms are available as supplemental material to this article and from the ETPLAS website.

Housing conditions are essential for ensuring animal welfare and high-quality research outcomes. In this study, we continuously monitored air quality—specifically ammonia, carbon dioxide, relative humidity, and temperature—in Individually Ventilated Cages (IVCs) housing five female or male C57BL/6N mice. The cages were cleaned either weekly or bi-weekly, and the data were collected as the mice aged from 100 to 348 days. The survival rate remained above 96%, with body weight increasing by 35–52% during the study period. The ammonia levels rose throughout the cleaning cycle, but averaged below 25 ppm. However, in the older, heavier mice with bi-weekly cage cleaning, the ammonia levels reached between 25 and 75 ppm, particularly in the males. While circadian rhythms influenced the ammonia concentration only to a small extent, the carbon dioxide levels varied between 800 and 3000 ppm, increasing by 30–50% at night and by 1000 ppm with body weight. Humidity also correlated primarily with the circadian rhythms (10% higher at night) and, to a lesser extent, with body weight, reaching ≥70% in the middle-aged mice. The temperature variations remained minimal, within a 1 °C range. We conclude that air quality assessments in IVCs should be conducted during animals’ active periods, and both housing density and biomass must be considered to optimise welfare.

Curdlan, a β-1,3/1,6-glucan found in Alcaligenes faecalis (A. faecalis) wall, activates innate and humoral immunity. The aim of this study is to evaluate whether pretreated rats with A. faecalis A12C could prevent sepsis disturbances and identify the immunomodulatory mechanisms involved. Experiments occurred in two stages: a survival study with 16 rats randomly divided into septic (SC) (n = 8) and septic pretreated (SA) (n = 8) groups and 45 rats divided into four groups: healthy (AGUSAN) (n = 9), septic (AGUIC) (n = 13), septic pretreated (AGUIA) (n = 14), and healthy pretreated (AGUSTO) (n = 9). Sepsis was induced by cecal ligation and puncture after 30 days of A. faecalis A12C pretreatment or without. SA group had a higher survival rate of 58% vs. 16% for SC group (P < 0.05). Overall, AGUIA showed better status than AGUIC (P < 0.01). Higher monocytosis was found in AGUIA and AGUSTO vs. AGUIC and AGUSAN, respectively (P < 0.05). A gradual increase in curdlan fecal concentration was observed in AGUIA during pretreatment. Fecal concentrations of Escherichia coli significantly decreased in AGUIA and AGUSTO. Bacterial load in urine, peritoneal lavage fluid (PLF), and bronchoalveolar lavage fluid (BALF) decreased (P < 0.05) in AGUIA vs. AGUIC. Finally, lower inflammation was observed in serum, BALF, and PLF, with reduced IL-6, IL-10, IL-1β, and TNF-α, along with less damage in lungs and peritoneum in AGUIA vs. AGUIC. These findings suggest the connection between curdlan—produced by A. faecalis A12C—with the immune system and the reduction in severity of experimental sepsis.

Demonstrated competence in laboratory animal science (LAS) is a prerequisite in Directive 2010/63/EU to work with animals used in scientific procedures, as it is essential to increase animal welfare, improve the quality of science, promote the acceptability of animal research and meet the demands of free movement of personnel and scientific exchange. Although since 2010 there have been eight clear steps to achieving the required competence of personnel working with animals used in science, it is not uncommon to see documentation for individuals who have just completed an LAS course that contains only education and training elements (three steps), for which the status of competence in LAS is granted. Here, a simplified summary of how competence in LAS should be delivered in eight steps according to EU recommendations is presented.

A strain ofAlcaligenes faecalisA12C (A. faecalisA12C) isolated fromArgyrosomus regiusis a probiotic in fish. Previous experiments showed thatA. faecalisA12C had inhibitory effects on the growth of multidrug-resistant bacteria. We aimed to confirm whetherA. faecalisA12C is safe and has adequate intestinal colonization in experimental rats, and evaluate its efficacy in an animal model of peritonitis.We used 30 male rats, randomly divided into 6 groups (n = 5): three groups (HA7, HA15, HA30) receivedA. faecalisA12C in drinking water (6 × 10⁸ CFU/mL) for 7 days, and three control groups received drinking water only. All groups were evaluated at 7, 15, and 30 days. Survival afterA. faecalisA12C administration was 100% in all groups. Mild eosinophilia (1.5%,p < 0.01) and increased aspartate aminotransferase (86 IU/L,p < 0.05) were observed in HA7, followed by progressive normalization. No histological signs of organ injury were found. We observed significantE. colidecline in faeces, parallel to an increase inA. faecalisA12C at 7 days.E. colihad a tendency to recover initial values, whileA. faecalisA12C disappeared from the intestinal microbiota at 30 days. To evaluate its efficacy against peritonitis, we studied two additional groups of animals: IA group pretreated withA. faecalisA12C beforeE. coliintra-abdominal inoculation, and IC group inoculated with noA. faecalisA12C. We found an increase in C-reactive protein, alanine aminotransferase, urea, and eosinophils in IC animals when compared with IA. Peritonitis was more evident in IC than in IA animals. Our findings suggest thatA. faecalisA12C altered clinically relevant parameters in sepsis and was associated with a lesser spread of infection.

Intestinal permeability (IP) tests are used to assess intestinal damage in patients and research models. The probe iohexol has shown advantages compared to ⁵¹Cr-EDTA or absorbable/nonabsorbable sugars. During IP tests, animals are housed in metabolic cages (MCs) to collect urine. We examined the performance of an iohexol IP test in mice. Rag1-/- (C57BL/6) mice of both sexes were divided into controls or treatment groups, the latter receiving injections of effector/memory T cells to induce intestinal inflammation. After two, four and five weeks (W), a single dose of iohexol was orally administered. Urine was collected seven times over 24 h in MCs. Iohexol concentration was measured by ELISA. Intestinal histological damage was scored in duodenal sections. In control and treated mice of both sexes, urinary excretion of iohexol peaked at 4 h. From W2 to W4/W5, urinary iohexol excretion increased in treated mice of both sexes, consistent with development of duodenitis in this model. Positive correlations were observed between the urinary excretion of iohexol in W4/W5 and the histological severity of duodenitis in treated male mice. We conclude that a 6 h cumulative urine sample appears sufficient to evaluate small IP to iohexol in this mouse model, improving animal welfare by reducing cage periods.

Typical features of celiac disease are small-bowel villus atrophy, crypt hyperplasia, and inflammation which develop gradually concomitant with ingestion of gluten. In addition, patients have anti-transglutaminase 2 (TG2) autoantibodies in their serum and tissues. The aim of this study was to establish whether celiac disease can be passively transferred to mice by serum or immunoglobulins. Serum aliquots or purified immunoglobulins (Ig) were intraperitoneally injected into Hsd:Athymic Nude-Foxn1nu mice for 8 or 27 days. As mice do not have proper IgA transport from peritoneum to blood, sera with a high content of IgG class anti-TG2 antibodies from untreated IgA-deficient celiac patients were used. Mouse sera were tested for celiac disease-specific autoantibodies, and several tissues were analyzed for autoantibody deposits targeted to TG2. Morphological assessment was made of the murine small intestinal mucosa. Injection of celiac disease patient sera or total IgG led to a significant delay in weight gain and mild diarrhea in a subset of mice. The mice injected with celiac patient sera or IgG had significantly decreased villus height crypt depth (Vh/CrD) ratios and celiac disease-specific autoantibody deposits targeted to TG2 in several tissues, including the small intestine. None of these features were observed in control mice. We conclude that administration of IgA-deficient celiac disease patient serum or total IgG induces both deterioration of the intestinal mucosa and clinical features of celiac disease in mice. The experimentally induced condition in the mice injected with patient serum or IgG resembles early developing celiac disease in humans. Celiac disease patient sera or total IgG was injected into athymic mice. A significant delay in weight gain and mild diarrhea was observed. Mice evinced significantly decreased villus height crypt depth ratios. Celiac disease-specific autoantibody deposits were present in several tissues. The condition in mice resembles early stage celiac disease in humans.