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3 pages, 187 KiB

Open AccessEditorial

Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice

by Garikoitz Azkona

Animals 2023, 13(19), 3063; https://doi.org/10.3390/ani13193063 - 29 Sep 2023

Cited by 1 | Viewed by 1635

Abstract

The regulatory framework for the use of animals in research in many countries is based on the 3Rs: replacement, reduction, and refinement [...] Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

Research

Jump to: Editorial, Review, Other

13 pages, 884 KiB

Open AccessArticle

Progress towards the Replacement of the Rabbit Blood Sugar Test for the Quantitative Determination of the Biological Activity of Insulins (USP <121>) with an In Vitro Assay

by Sabrina Rüggeberg, Antje Wanglin, Özlem Demirel, Rüdiger Hack, Birgit Niederhaus, Bernd Bidlingmaier, Matthias Blumrich and Dirk Usener

Animals 2023, 13(18), 2953; https://doi.org/10.3390/ani13182953 - 18 Sep 2023

Viewed by 1836

Abstract

For the quantification of insulin activity, United States Pharmacopeia (USP) general chapter <121> continues to require the rabbit blood sugar test. For new insulin or insulin analogue compounds, those quantitative data are expected for stability or comparability studies. At Sanofi, many rabbits were [...] Read more.

For the quantification of insulin activity, United States Pharmacopeia (USP) general chapter <121> continues to require the rabbit blood sugar test. For new insulin or insulin analogue compounds, those quantitative data are expected for stability or comparability studies. At Sanofi, many rabbits were used to fulfil the authority’s requirements to obtain quantitative insulin bioactivity data until the in vivo test was replaced. In order to demonstrate comparability between the in vivo and in vitro test systems, this study was designed to demonstrate equivalency. The measurement of insulin lispro and insulin glargine drug substance and drug product batches, including stress samples (diluted or after temperature stress of 30 min at 80 °C), revealed a clear correlation between the in vitro and in vivo test results. The recovery of quantitative in vitro in-cell Western (ICW) results compared to the in vivo test results was within the predefined acceptance limits of 80% to 125%. Thus, the in vitro ICW cell-based bioassay leads to results that are equivalent to the rabbit blood sugar test per USP <121>, and it is highly suitable for insulin activity quantification. For future development compounds, the in vitro in-cell Western cell-based assay can replace the rabbit blood sugar test required by USP <121>. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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12 pages, 1563 KiB

Open AccessCommunication

Acclimation and Blood Sampling: Effects on Stress Markers in C57Bl/6J Mice

by Nerea Marin, Amparo Moragon, Domingo Gil, Francisco Garcia-Garcia and Viviana Bisbal

Animals 2023, 13(18), 2816; https://doi.org/10.3390/ani13182816 - 5 Sep 2023

Cited by 3 | Viewed by 1856

Abstract

Blood sampling in rodents is common practice in scientific studies. Some of the refined methods widely used are the puncture of the saphenous vein or tail vein, or even tail docking. The handling needs of these different blood sampling methods are different and [...] Read more.

Blood sampling in rodents is common practice in scientific studies. Some of the refined methods widely used are the puncture of the saphenous vein or tail vein, or even tail docking. The handling needs of these different blood sampling methods are different and can directly affect stress, increasing the variability of the study. Moreover, there is less aversion and stress if the animal is accustomed to the environment, handling and technique. Therefore, our study aimed to assess the influence of these three blood sampling techniques (saphenous puncture, tail vein puncture and tail vein docking) and the use of previous acclimation on different indicators of animal stress, assessing blood glucose concentrations and faecal corticosterone metabolites (FCMs). Twenty-four young adult male and female C57Bl6/J mice were divided in three groups by sampling method: tail docking (TD), saphenous vein puncture (SV) and caudal vein puncture (CV) groups. All mice were studied with and without acclimation, which was performed during 9 consecutive days. The results showed that both males and females present very similar responses to the different handling and sampling methods without significant differences. Nevertheless, acclimation in all sampling methods decreased glucose and FCM levels significantly. The method that obtained the lowest glucose and FCM levels with significance was saphenous vein puncture. Therefore, we can say that it causes less stress when performing prior acclimation, even when this involves greater handling of the animal. Our results contribute to refinement within the 3R concept and could serve researchers to programme and select a good handling technique and a welfare-friendly blood sampling method for their experiments. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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25 pages, 4944 KiB

Open AccessArticle

Refining Stereotaxic Neurosurgery Techniques and Welfare Assessment for Long-Term Intracerebroventricular Device Implantation in Rodents

by Ester Pérez-Martín, Almudena Coto-Vilcapoma, Juan Castilla-Silgado, María Rodríguez-Cañón, Catuxa Prado, Gabriel Álvarez, Marco Antonio Álvarez-Vega, Benjamín Fernández-García, Manuel Menéndez-González and Cristina Tomás-Zapico

Animals 2023, 13(16), 2627; https://doi.org/10.3390/ani13162627 - 14 Aug 2023

Viewed by 4485

Abstract

Stereotaxic surgeries enable precise access to specific brain regions, being of particular interest for chronic intracerebroventricular drug delivery. However, the challenge of long-term studies at this level is to allow the implantation of drug storage devices and their correct intrathecal connection while guaranteeing [...] Read more.

Stereotaxic surgeries enable precise access to specific brain regions, being of particular interest for chronic intracerebroventricular drug delivery. However, the challenge of long-term studies at this level is to allow the implantation of drug storage devices and their correct intrathecal connection while guaranteeing animal welfare during the entire study period. In this study, we propose an optimized method for safe intrathecal device implantation, focusing on preoperative, intraoperative, and postoperative procedures, following the 3Rs principle and animal welfare regulations. Our optimized protocol introduces three main refinements. Firstly, we modify the dimensions of the implantable devices, notably diminishing the device-to-mouse weight ratio. Secondly, we use a combination of cyanoacrylate tissue adhesive and UV light-curing resin, which decreases surgery time, improves healing, and notably minimizes cannula detachment or adverse effects. Thirdly, we develop a customized welfare assessment scoresheet to accurately monitor animal well-being during long-term implantations. Taken together, these refinements positively impacted animal welfare by minimizing the negative effects on body weight, surgery-related complications, and anxiety-like behaviors. Overall, the proposed refinements have the potential to reduce animal use, enhance experimental data quality, and improve reproducibility. Additionally, these improvements can be extended to other neurosurgical techniques, thereby advancing neuroscience research, and benefiting the scientific community. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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16 pages, 933 KiB

Open AccessArticle

Rehoming and Other Refinements and Replacement in Procedures Using Golden Hamsters in SARS-CoV-2 Vaccine Research

by Malan Štrbenc, Urška Kuhar, Duško Lainšček, Sara Orehek, Brigita Slavec, Uroš Krapež, Tadej Malovrh and Gregor Majdič

Animals 2023, 13(16), 2616; https://doi.org/10.3390/ani13162616 - 14 Aug 2023

Viewed by 1617

Abstract

Effective vaccines are needed to fight the COVID-19 pandemic. Forty golden hamsters were inoculated with two promising vaccine candidates and eighteen animals were used in pilot trials with viral challenge. ELISA assays were performed to determine endpoint serum titres for specific antibodies and [...] Read more.

Effective vaccines are needed to fight the COVID-19 pandemic. Forty golden hamsters were inoculated with two promising vaccine candidates and eighteen animals were used in pilot trials with viral challenge. ELISA assays were performed to determine endpoint serum titres for specific antibodies and virus neutralisation tests were used to evaluate the efficacy of antibodies. All tests with serum from vaccinated hamsters were negative even after booster vaccinations and changes in vaccination protocol. We concluded that antibodies did not have sufficient neutralising properties. Refinements were observed at all steps, and the in vitro method (virus neutralisation test) presented a replacement measure and ultimately lead to a reduction in the total number of animals used in the project. The institutional animal welfare officer and institutional designated veterinarian approved the reuse or rehoming of the surplus animals. Simple socialization procedures were performed and ultimately 19 animals were rehomed, and feedback was collected. Recently, FELASA published recommendations for rehoming of animals used for scientific and educational purposes, with species-specific guidelines, including mice, rats, and rabbits. Based on our positive experience and feedback from adopters, we concluded that the rehoming of rodents, including hamsters, is not only possible, but highly recommended. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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14 pages, 587 KiB

Open AccessArticle

Severity Classification of Laboratory Animal Procedures in Two Belgian Academic Institutions

by Stéphanie De Vleeschauwer, Kathleen Lambaerts, Sophie Hernot and Karlijn Debusschere

Animals 2023, 13(16), 2581; https://doi.org/10.3390/ani13162581 - 10 Aug 2023

Cited by 1 | Viewed by 2298

Abstract

According to the EU Directive 2010/63, all animal procedures must be classified as non-recovery, mild, moderate or severe. Several examples are included in the Directive to help in severity classification. Since the implementation of the Directive, different publications and guidelines have been disseminated [...] Read more.

According to the EU Directive 2010/63, all animal procedures must be classified as non-recovery, mild, moderate or severe. Several examples are included in the Directive to help in severity classification. Since the implementation of the Directive, different publications and guidelines have been disseminated on the topic. However, due to the large variety of disease models and animal procedures carried out in many different animal species, guidance on the severity classification of specific procedures or models is often lacking or not specific enough. The latter is especially the case in disease models where the level of pain, suffering, distress and lasting harm depends on the duration of the study (for progressive disease models) or the dosage given (for infectious or chemically induced disease models). This, in turn, may lead to inconsistencies in severity classification between countries, within countries and even within institutions. To overcome this, two Belgian academic institutions with a focus on biomedical research collaborated to develop a severity classification for all the procedures performed. This work started with listing all in-house procedures and assigning them to 16 (sub)categories. First, we determined which parameters, such as clinical signs, dosage or duration, were crucial for severity classification within a specific (sub)category. Next, a severity classification was assigned to the different procedures, which was based on professional judgment by the designated veterinarians, members of the animal welfare body (AWB) and institutional animal ethics committee (AEC), integrating the available literature and guidelines. During the classification process, the use of vague terminology, such as ‘minor impact’, was avoided as much as possible. Instead, well-defined cut-offs between severity levels were used. Furthermore, we sought to define common denominators to group procedures and to be able to classify new procedures more easily. Although the primary aim is to address prospective severity, this can also be used to assess actual severity. In summary, we developed a severity classification for all procedures performed in two academic, biomedical institutions. These include many procedures and disease models in a variety of animal species for which a severity classification was not reported so far, or the terms that assign them to a different severity were too vague. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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15 pages, 4310 KiB

Open AccessArticle

Camera-Based Respiration Monitoring of Unconstrained Rodents

by Lukas Breuer, Lucas Mösch, Janosch Kunczik, Verena Buchecker, Heidrun Potschka, Michael Czaplik and Carina Barbosa Pereira

Animals 2023, 13(12), 1901; https://doi.org/10.3390/ani13121901 - 7 Jun 2023

Cited by 1 | Viewed by 2277

Abstract

Animal research has always been crucial for various medical and scientific breakthroughs, providing information on disease mechanisms, genetic predisposition to diseases, and pharmacological treatment. However, the use of animals in medical research is a source of great controversy and ongoing debate in modern [...] Read more.

Animal research has always been crucial for various medical and scientific breakthroughs, providing information on disease mechanisms, genetic predisposition to diseases, and pharmacological treatment. However, the use of animals in medical research is a source of great controversy and ongoing debate in modern science. To ensure a high level of bioethics, new guidelines have been adopted by the EU, implementing the 3R principles to replace animal testing wherever possible, reduce the number of animals per experiment, and refine procedures to minimize stress and pain. Supporting these guidelines, this article proposes an improved approach for unobtrusive, continuous, and automated monitoring of the respiratory rate of laboratory rats. It uses the cyclical expansion and contraction of the rats’ thorax/abdominal region to determine this physiological parameter. In contrast to previous work, the focus is on unconstrained animals, which requires the algorithms to be especially robust to motion artifacts. To test the feasibility of the proposed approach, video material of multiple rats was recorded and evaluated. High agreement was obtained between RGB imaging and the reference method (respiratory rate derived from electrocardiography), which was reflected in a relative error of 5.46%. The current work shows that camera-based technologies are promising and relevant alternatives for monitoring the respiratory rate of unconstrained rats, contributing to the development of new alternatives for a continuous and objective assessment of animal welfare, and hereby guiding the way to modern and bioethical research. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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13 pages, 1885 KiB

Open AccessArticle

Evaluation of Parameters Which Influence Voluntary Ingestion of Supplements in Rats

by Santiago Ruvira, Pilar Rodríguez-Rodríguez, Silvia Cañas, David Ramiro-Cortijo, Yolanda Aguilera, David Muñoz-Valverde and Silvia M. Arribas

Animals 2023, 13(11), 1827; https://doi.org/10.3390/ani13111827 - 31 May 2023

Cited by 7 | Viewed by 1904

Abstract

Drug safety and efficacy studies frequently use oral gavage, but repetitive usage may cause problems. Administration through voluntary ingestion represents an opportunity for refinement. We aimed to develop a protocol for voluntary ingestion of gelatin-based supplements in rats, assessing the influence of age, [...] Read more.

Drug safety and efficacy studies frequently use oral gavage, but repetitive usage may cause problems. Administration through voluntary ingestion represents an opportunity for refinement. We aimed to develop a protocol for voluntary ingestion of gelatin-based supplements in rats, assessing the influence of age, sex, fasting (4 h), and additives (vanilla, VF; sucralose, S), and to test it in lactating dams. Three-week-old and 5-month-old Sprague-Dawley rats were placed individually in an empty cage containing a gelatin cube and trained daily (5 days/week), recording the day the whole cube was consumed (latency). Rats trained prior to gestation were offered a gelatin containing 250 mg/kg cocoa shell extract (CSE) during lactation. Rats that did not eat the cube after 8 training days were considered non-habituated, with a proportion similar in young males (7.1%), young females (11.1%), and adult females (10.3%), but significantly higher in adult males (39.3%). Excluding non-habituated rats, latency was 2–3 days, without differences between young and adult rats (p = 0.657) or between males and females (p = 0.189). VF or VF + S in the gelatin did not modify latency, while fasting significantly reduced it in females (p = 0.007) but not in males (p = 0.501). During lactation, trained females ate the CSE-gelatin within 1–5 min without litter problems. Conclusions: Acceptance of a gelatin-based supplement is negatively influenced by male sex, facilitated by fasting, and not modified by additives. Training is remembered after 2 months and does not interfere with lactation. Gelatin-based voluntary ingestion is suitable to administer drugs that need to pass through the digestive system, ensuring adequate dosage, and is important to detect non-habituated rats prior to the study. The current protocol may be implemented by training the rats in their own cage. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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12 pages, 2797 KiB

Open AccessArticle

Longitudinal Studies on Alzheimer Disease Mouse Models with Multiple Tracer PET/CT: Application of Reduction and Refinement Principles in Daily Practice to Safeguard Animal Welfare during Progressive Aging

by Giovanna Palumbo, Lea Helena Kunze, Rosel Oos, Karin Wind-Mark, Simon Lindner, Barbara von Ungern-Sternberg, Peter Bartenstein, Sibylle Ziegler and Matthias Brendel

Animals 2023, 13(11), 1812; https://doi.org/10.3390/ani13111812 - 30 May 2023

Cited by 2 | Viewed by 1791

Abstract

Longitudinal studies on mouse models related to Alzheimer disease (AD) pathology play an important role in the investigation of therapeutic targets to help pharmaceutical research in the development of new drugs and in the attempt of an early diagnosis that can contribute to [...] Read more.

Longitudinal studies on mouse models related to Alzheimer disease (AD) pathology play an important role in the investigation of therapeutic targets to help pharmaceutical research in the development of new drugs and in the attempt of an early diagnosis that can contribute to improving people’s quality of life. There are several advantages to enriching longitudinal studies in AD models with Positron Emission Tomography (PET); among these advantages, the possibility of following the principle of the 3Rs of animal welfare is fundamental. In this manuscript, good daily experimental practice focusing on animal welfare is described and commented upon, based on the experience attained from studies conducted in our Nuclear Medicine department. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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16 pages, 5301 KiB

Open AccessArticle

Studying and Analyzing Humane Endpoints in the Fructose-Fed and Streptozotocin-Injected Rat Model of Diabetes

by Rita Silva-Reis, Ana I. Faustino-Rocha, Jéssica Silva, Abigaël Valada, Tiago Azevedo, Lara Anjos, Lio Gonçalves, Maria de Lurdes Pinto, Rita Ferreira, Artur M. S. Silva, Susana M. Cardoso and Paula A. Oliveira

Animals 2023, 13(8), 1397; https://doi.org/10.3390/ani13081397 - 18 Apr 2023

Cited by 5 | Viewed by 2824

Abstract

This work aimed to define a humane endpoint scoring system able to objectively identify signs of animal suffering in a rat model of type 2 diabetes. Sprague-Dawley male rats were divided into control and induced group. The induced animals drink a 10% fructose [...] Read more.

This work aimed to define a humane endpoint scoring system able to objectively identify signs of animal suffering in a rat model of type 2 diabetes. Sprague-Dawley male rats were divided into control and induced group. The induced animals drink a 10% fructose solution for 14 days. Then, received an administration of streptozotocin (40 mg/kg). Animals’ body weight, water and food consumption were recorded weekly. To evaluate animal welfare, a score sheet with 14 parameters was employed. Blood glucose levels were measured at three time points. After seven weeks of initiating the protocol, the rats were euthanized. The induced animals showed weight loss, polyuria, polyphagia, and polydipsia. According to our humane endpoints table, changes in animal welfare became noticeable after the STZ administration. None of the animals hit the critical score limit (four). Data showed that the most effective parameters to assess welfare in this type 2 diabetes rat induction model were dehydration, grooming, posture, abdominal visualization, and stool appearance. The glycemia was significantly higher in the induced group when compared to the controls (p < 0.01). Induced animals’ murinometric and nutritional parameters were significantly lower than the controls (p < 0.01). Our findings suggest that in this rat model of type 2 diabetes with STZ-induced following fructose consumption, our list of humane endpoints is suitable for monitoring the animals’ welfare. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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12 pages, 953 KiB

Open AccessArticle

Individualized Housing Modifies the Immune–Endocrine System in CD1 Adult Male Mice

by Iván Ortega-Saez, Alina Díez-Solinska, Roger Grífols, Cristina Martí, Carolina Zamora, Maider Muñoz-Culla, Oscar Vegas and Garikoitz Azkona

Animals 2023, 13(6), 1026; https://doi.org/10.3390/ani13061026 - 10 Mar 2023

Cited by 3 | Viewed by 2190

Abstract

In the last years, different research groups have made considerable efforts to improve the care and use of animals in research. Mice (Mus musculus) are the most widely used animal species in research in the European Union and are sociable and [...] Read more.

In the last years, different research groups have made considerable efforts to improve the care and use of animals in research. Mice (Mus musculus) are the most widely used animal species in research in the European Union and are sociable and hierarchical creatures. During experiments, researchers tend to individualize males, but no consideration is given to whether this social isolation causes them stress. The aim of this study was, therefore, to explore whether 4 weeks of social isolation could induce changes in different physiological parameters in adult Crl:CD1(ICR) (CD1) males, which may interfere with experimental results. Body weight, blood cells, and fecal corticosterone metabolites levels were the analyzed parameters. Blood and fecal samples were collected at weeks 1 and 4 of the experimental procedure. Four weeks of single housing produced a significant time-dependent decrease in monocytes and granulocytes. Fecal corticosterone metabolite levels were higher in single-housed mice after 1 week and then normalized after 4 weeks of isolation. Body weight, red blood cells, and platelets remained unchanged in both groups during this period. We can, therefore, conclude that social isolation affects some immune and endocrine parameters, and that this should be taken into account in the interpretation of research data. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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12 pages, 1941 KiB

Open AccessArticle

Are Currently Selected Laboratory Animals Useful in the Research of How Female Hormones Influence Orthodontic Biomechanics?

by Małgorzata Peruga, Beata Kawala, Michał Sarul, Jakub Kotowicz and Joanna Lis

Animals 2023, 13(4), 629; https://doi.org/10.3390/ani13040629 - 10 Feb 2023

Cited by 3 | Viewed by 2354

Abstract

Animal testing was and remains the only method of introducing a certain treatment and medical procedure on humans. On the other hand, animals have their rights resulting from applicable legal acts, including Directive 2010/63/EU and, indirectly, the World Medical Association International Code of [...] Read more.

Animal testing was and remains the only method of introducing a certain treatment and medical procedure on humans. On the other hand, animals have their rights resulting from applicable legal acts, including Directive 2010/63/EU and, indirectly, the World Medical Association International Code of Medical Ethics (Helsinki Declaration, 1975, amended 2000). Thus, the question arises whether the credibility of the results of hormonal and orthodontic tests obtained so far and their usefulness for the human population is scientifically justified and worth sacrificing laboratory animals for. Especially that, according to statistical data, about 50% of laboratory animals are euthanized at the conclusion of the experiments. The aim of this article was to determine whether animal experiments are scientifically or morally justified in bringing significant evidence in studies that may validate the influence of changes in the concentration of female hormones secreted by the ovaries in various phases of the menstrual cycle in young patients on the duration of an increased tooth movement rate in orthodontic treatment. Papers reporting the results of the original research into female hormones, either natural or exogeneous ones, likely to alternate the orthodontic tooth movement rate were critically evaluated in terms of animal selection. Thorough analysis supported by veterinary knowledge proved that none of the publications enabled an extrapolation of the results to humans. The evaluation of the relation between the rate of tooth movement upon loading with orthodontic forces and hormones either secreted during the menstrual cycle of women or released from the contraceptives already present in the market, does not require sacrificing laboratory animals. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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11 pages, 11842 KiB

Open AccessCommunication

Stress Evaluation of Mouse Husbandry Environments for Improving Laboratory Animal Welfare

by Gwang-Hoon Lee, KilSoo Kim and Woori Jo

Animals 2023, 13(2), 249; https://doi.org/10.3390/ani13020249 - 10 Jan 2023

Cited by 9 | Viewed by 3333

Abstract

Animal welfare is recognized as essential for the coexistence of humans and animals. Considering the increased demand and interest in animal welfare, many methods for improving animal welfare are being devised, but which method reduces animal stress has not been scientifically verified. Therefore, [...] Read more.

Animal welfare is recognized as essential for the coexistence of humans and animals. Considering the increased demand and interest in animal welfare, many methods for improving animal welfare are being devised, but which method reduces animal stress has not been scientifically verified. Therefore, reducing animal stress by providing a proper breeding environment and environmental enrichment can be the basis for animal study. In this study, stress levels were assessed based on the mouse-breeding environment. We considered that the higher the body weight and the lower the corticosterone concentration, the lower the stress. According to the results, animals in the individual ventilation cages were determined to have lower serum cortisol concentrations, while the body weight of the animals was increased when in individual ventilation cages compared with individual isolated cages and when providing environmental enrichment compared with group breeding or not providing environmental enrichment. The results provide appropriate guidelines for improving laboratory animal welfare. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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10 pages, 1284 KiB

Open AccessArticle

Clicker Training Mice for Improved Compliance in the Catwalk Test

by Jana Dickmann, Fernando Gonzalez-Uarquin, Sandra Reichel, Dorothea Pichl, Konstantin Radyushkin, Jan Baumgart and Nadine Baumgart

Animals 2022, 12(24), 3545; https://doi.org/10.3390/ani12243545 - 15 Dec 2022

Cited by 2 | Viewed by 2392

Abstract

The CatWalk test relies on the run of mice across the platform to measure a constant speed with low variation. Mice usually require a stimulus to walk to the end of the catwalk. However, such stimuli are usually aversive and can impair welfare. [...] Read more.

The CatWalk test relies on the run of mice across the platform to measure a constant speed with low variation. Mice usually require a stimulus to walk to the end of the catwalk. However, such stimuli are usually aversive and can impair welfare. Positive reinforcement training of laboratory animals is a thriving tool for refinement and contributes to meeting the demands instituted by Directive 2010/63/EU. We have already demonstrated the positive effects of clicker training. In this study, we trained male and female mice to complete the CatWalk protocol while assessing the effects of training on their well-being (Open Filed and Elevated Plus Maze). In the CatWalk test, we observed that clicker training improved the running speed of the mice. In addition, clicker training reduced the number of runs required by mice, which was more pronounced in males. Clicker training lowered anxiety-like behaviors in our mice, especially in females, where a significant difference was observed between trained and untrained ones. Based on our findings, we hypothesize that clicker training is an effective tool to motivate mice and increase performance on the CatWalk test without potentially impairing their welfare (e.g., by puffing them). Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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13 pages, 6276 KiB

Open AccessArticle

Brain Organoids to Evaluate Cellular Therapies

by Ana Belén García-Delgado, Rafael Campos-Cuerva, Cristina Rosell-Valle, María Martin-López, Carlos Casado, Daniela Ferrari, Javier Márquez-Rivas, Rosario Sánchez-Pernaute and Beatriz Fernández-Muñoz

Animals 2022, 12(22), 3150; https://doi.org/10.3390/ani12223150 - 15 Nov 2022

Cited by 6 | Viewed by 3902

Abstract

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the [...] Read more.

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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Review

Jump to: Editorial, Research, Other

16 pages, 634 KiB

Open AccessReview

Unmasking the Adverse Impacts of Sex Bias on Science and Research Animal Welfare

by Elizabeth A. Nunamaker and Patricia V. Turner

Animals 2023, 13(17), 2792; https://doi.org/10.3390/ani13172792 - 2 Sep 2023

Cited by 5 | Viewed by 5702

Abstract

Sex bias in biomedical and natural science research has been prevalent for decades. In many cases, the female estrous cycle was thought to be too complex an issue to model for, and it was thought to be simpler to only use males in [...] Read more.

Sex bias in biomedical and natural science research has been prevalent for decades. In many cases, the female estrous cycle was thought to be too complex an issue to model for, and it was thought to be simpler to only use males in studies. At times, particularly when studying efficacy and safety of new therapeutics, this sex bias has resulted in over- and under-medication with associated deleterious side effects in women. Many sex differences have been recognized that are unrelated to hormonal variation occurring during the estrous cycle. Sex bias also creates animal welfare challenges related to animal over-production and wastage, insufficient consideration of welfare (and scientific) impact related to differential housing of male vs female animals within research facilities, and a lack of understanding regarding differential requirements for pain recognition and alleviation in male versus female animals. Although many funding and government agencies require both sexes to be studied in biomedical research, many disparities remain in practice. This requires further enforcement of expectations by the Institutional Animal Care and Use Committee when reviewing protocols, research groups when writing grants, planning studies, and conducting research, and scientific journals and reviewers to ensure that sex bias policies are enforced. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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32 pages, 931 KiB

Open AccessReview

The 3Rs in Experimental Liver Disease

by Sebastian Martinez-Lopez, Enrique Angel-Gomis, Elisabet Sanchez-Ardid, Alberto Pastor-Campos, Joanna Picó and Isabel Gomez-Hurtado

Animals 2023, 13(14), 2357; https://doi.org/10.3390/ani13142357 - 19 Jul 2023

Cited by 3 | Viewed by 2907

Abstract

Patients with cirrhosis present multiple physiological and immunological alterations that play a very important role in the development of clinically relevant secondary complications to the disease. Experimentation in animal models is essential to understand the pathogenesis of human diseases and, considering the high [...] Read more.

Patients with cirrhosis present multiple physiological and immunological alterations that play a very important role in the development of clinically relevant secondary complications to the disease. Experimentation in animal models is essential to understand the pathogenesis of human diseases and, considering the high prevalence of liver disease worldwide, to understand the pathophysiology of disease progression and the molecular pathways involved, due to the complexity of the liver as an organ and its relationship with the rest of the organism. However, today there is a growing awareness about the sensitivity and suffering of animals, causing opposition to animal research among a minority in society and some scientists, but also about the attention to the welfare of laboratory animals since this has been built into regulations in most nations that conduct animal research. In 1959, Russell and Burch published the book “The Principles of Humane Experimental Technique”, proposing that in those experiments where animals were necessary, everything possible should be done to try to replace them with non-sentient alternatives, to reduce to a minimum their number, and to refine experiments that are essential so that they caused the least amount of pain and distress. In this review, a comprehensive summary of the most widely used techniques to replace, reduce, and refine in experimental liver research is offered, to assess the advantages and weaknesses of available experimental liver disease models for researchers who are planning to perform animal studies in the near future. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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24 pages, 1365 KiB

Open AccessReview

The Importance of Animal Models in Biomedical Research: Current Insights and Applications

by Adriana Domínguez-Oliva, Ismael Hernández-Ávalos, Julio Martínez-Burnes, Adriana Olmos-Hernández, Antonio Verduzco-Mendoza and Daniel Mota-Rojas

Animals 2023, 13(7), 1223; https://doi.org/10.3390/ani13071223 - 31 Mar 2023

Cited by 60 | Viewed by 31495

Abstract

Animal research is considered a key element in advance of biomedical science. Although its use is controversial and raises ethical challenges, the contribution of animal models in medicine is essential for understanding the physiopathology and novel treatment alternatives for several animal and human [...] Read more.

Animal research is considered a key element in advance of biomedical science. Although its use is controversial and raises ethical challenges, the contribution of animal models in medicine is essential for understanding the physiopathology and novel treatment alternatives for several animal and human diseases. Current pandemics’ pathology, such as the 2019 Coronavirus disease, has been studied in primate, rodent, and porcine models to recognize infection routes and develop therapeutic protocols. Worldwide issues such as diabetes, obesity, neurological disorders, pain, rehabilitation medicine, and surgical techniques require studying the process in different animal species before testing them on humans. Due to their relevance, this article aims to discuss the importance of animal models in diverse lines of biomedical research by analyzing the contributions of the various species utilized in science over the past five years about key topics concerning human and animal health. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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20 pages, 15858 KiB

Open AccessFeature PaperReview

Ultrasound Guided Surgery as a Refinement Tool in Oncology Research

by Juan Antonio Camara Serrano

Animals 2022, 12(23), 3445; https://doi.org/10.3390/ani12233445 - 6 Dec 2022

Cited by 2 | Viewed by 3386

Abstract

Refinement is one of the ethical pillars of the use of animals in research. Ultrasonography is currently used in human medicine as a surgical tool for guided biopsies and this idea can be applied to preclinical research thanks to the development of specific [...] Read more.

Refinement is one of the ethical pillars of the use of animals in research. Ultrasonography is currently used in human medicine as a surgical tool for guided biopsies and this idea can be applied to preclinical research thanks to the development of specific instruments. This will eliminate the necessity of a surgical opening for implanting cells in specific organs or taking samples from tissues. The approach for the injection will depend on the target but most of the case is going to be lateral, with the probe in a ventral position and the needle going into from the lateral. This is the situation for the thyroid gland, heart, liver, spleen, kidney, pancreas, uterus, and testicles. Other approaches, such as the dorsal, can be used in the spleen or kidney. The maximum injected volume will depend on the size of the structure. For biopsies, the technical protocol is similar to the injection knowing that in big organs such as the liver, spleen, or kidney we can take several samples moving slightly the needle inside the structure. In all cases, animals must be anesthetized and minimum pain management is required after the intervention. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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25 pages, 6760 KiB

Open AccessReview

Challenges with Assessing and Treating Pain in Research Primates: A Focused Survey and Literature Review

by Emilie A. Paterson and Patricia V. Turner

Animals 2022, 12(17), 2304; https://doi.org/10.3390/ani12172304 - 5 Sep 2022

Cited by 8 | Viewed by 3461

Abstract

Research primates may undergo surgical procedures making effective pain management essential to ensure good animal welfare and unbiased scientific data. Adequate pain mitigation is dependent on whether veterinarians, technicians, researchers, and caregivers can recognize and assess pain, as well as the availability of [...] Read more.

Research primates may undergo surgical procedures making effective pain management essential to ensure good animal welfare and unbiased scientific data. Adequate pain mitigation is dependent on whether veterinarians, technicians, researchers, and caregivers can recognize and assess pain, as well as the availability of efficacious therapeutics. A survey was conducted to evaluate primate veterinary approaches to pain assessment and alleviation, as well as expressed challenges for adequately managing primate pain. The survey (n = 93 respondents) collected information regarding institutional policies and procedures for pain recognition, methods used for pain relief, and perceived levels of confidence in primate pain assessment. Results indicated that 71% (n = 60) of respondents worked at institutions that were without formal experimental pain assessment policies. Pain assessment methods were consistent across respondents with the majority evaluating pain based on changes in general activity levels (100%, n = 86) and food consumption (97%, n = 84). Self-reported confidence in recognizing and managing pain ranged from slightly confident to highly confident, and there was a commonly expressed concern about the lack of objective pain assessment tools and science-based evidence regarding therapeutic recommendations of analgesics for research primates. These opinions correspond with significant gaps in the primate pain management literature, including limited specific pharmacokinetic data and efficacy testing for commonly used analgesics in research primate species as well as limited research on objective and specific measures of pain in research primates. These results demonstrate that there are inconsistencies in institutional policies and procedures surrounding pain management in research primates and a lack of objective pain assessment methods. Demonstrating the gaps and challenges in primate pain management can inform guideline development and suggest areas for future research. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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11 pages, 254 KiB

Open AccessConcept Paper

A Model Framework for the Estimation of Animal ‘Suffering’: Its Use in Predicting and Retrospectively Assessing the Impact of Experiments on Animals

by David B. Morton

Animals 2023, 13(5), 800; https://doi.org/10.3390/ani13050800 - 22 Feb 2023

Cited by 5 | Viewed by 2654

Abstract

This paper presents and illustrates, with a working example, a hypothesis for the assessment of ongoing severity before and during an experiment that will enable humane endpoints and intervention points to be applied accurately and reproducibly, as well as helping to implement any [...] Read more.

This paper presents and illustrates, with a working example, a hypothesis for the assessment of ongoing severity before and during an experiment that will enable humane endpoints and intervention points to be applied accurately and reproducibly, as well as helping to implement any national legal severity limits in subacute and chronic animal experiments, e.g., as specified by the competent authority. The underlying assumption of the model framework is that the degree of deviation from normality of specified measurable biological criteria will reflect the level of pain, suffering, distress and lasting harm incurred by or during an experiment. The choice of criteria will normally reflect the impact on an animal and have to be chosen by scientists and those caring for the animals. They will usually include measurements of good health such as temperature, body weight, body condition and behaviour, which vary according to the species, husbandry and experimental protocols and, in some species, unusual parameters such as time of the year (e.g., migrating birds). In animal research legislation, endpoints or severity limits may be specified so that individual animals do not suffer unnecessarily or endure severe pain and distress that is long-lasting (Directive 2010/63/EU, Art.15.2). In addition, the overall severity is estimated and classified as part of the harm: benefit licence assessment. I present a mathematical model to analyse the measurement data to determine the degree of harm (or severity) incurred. The results can be used to initiate alleviative treatment if required or if permitted during the course of an experiment. In addition, any animal determined to have breached the severity classification of a procedure can be humanely killed, treated or withdrawn from the experiment. The system incorporates the flexibility to be used in most animal research work by being tailored to the research, the procedures carried out and the species under investigation. The criteria used to score severity can also be used as additional scientific outcome criteria and for an analysis of the scientific integrity of the project. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

14 pages, 622 KiB

Open AccessCommentary

3Rs Principle and Legislative Decrees to Achieve High Standard of Animal Research

by Paolo Verderio, Mara Lecchi, Chiara Maura Ciniselli, Bjorn Shishmani, Giovanni Apolone and Giacomo Manenti

Animals 2023, 13(2), 277; https://doi.org/10.3390/ani13020277 - 13 Jan 2023

Cited by 16 | Viewed by 4224

Abstract

Animal experimentation is a vast ecosystem that tries to make different issues such as legislative, ethical and scientific coexist. Research in animal experimentation has made many strides thanks to the 3Rs principle and the attached legislative decrees, but for this very reason, it [...] Read more.

Animal experimentation is a vast ecosystem that tries to make different issues such as legislative, ethical and scientific coexist. Research in animal experimentation has made many strides thanks to the 3Rs principle and the attached legislative decrees, but for this very reason, it needs to be evenly implemented both among the countries that have adhered to the decrees and among the team members who design and execute the experimental practice. In this article, we emphasize the importance of the 3Rs principle’s application, with a particular focus on the concept of Reduction and related key aspects that can best be handled with the contribution of experts from different fields. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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19 pages, 1328 KiB

Open AccessSystematic Review

Aggression in Group-Housed Male Mice: A Systematic Review

by Elin M. Weber, Josefina Zidar, Birgit Ewaldsson, Kaisa Askevik, Eva Udén, Emma Svensk and Elin Törnqvist

Animals 2023, 13(1), 143; https://doi.org/10.3390/ani13010143 - 30 Dec 2022

Cited by 13 | Viewed by 5234

Abstract

Aggression among group-housed male mice is a major animal welfare concern often observed at animal facilities. Studies designed to understand the causes of male mice aggression have used different methodological approaches and have been heterogeneous, using different strains, environmental enrichments, housing conditions, group [...] Read more.

Aggression among group-housed male mice is a major animal welfare concern often observed at animal facilities. Studies designed to understand the causes of male mice aggression have used different methodological approaches and have been heterogeneous, using different strains, environmental enrichments, housing conditions, group formations and durations. By conducting a systematic literature review based on 198 observed conclusions from 90 articles, we showed that the methodological approach used to study aggression was relevant for the outcome and suggested that home cage observations were better when studying home cage aggression than tests provoking aggression outside the home cage. The study further revealed that aggression is a complex problem; one solution will not be appropriate for all animal facilities and all research projects. Recommendations were provided on promising tools to minimize aggression, based on the results, which included what type of environmental enrichments could be appropriate and which strains of male mice were less likely to be aggressive. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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13 pages, 1671 KiB

Open AccessSystematic Review

Refinement in the European Union: A Systematic Review

by Alina Díez-Solinska, Oscar Vegas and Garikoitz Azkona

Animals 2022, 12(23), 3263; https://doi.org/10.3390/ani12233263 - 23 Nov 2022

Cited by 10 | Viewed by 1903

Abstract

Refining experiments and housing conditions so as to cause the minimum possible pain and distress is one of the three principles (3Rs) on which Directive 2010/63/EU is based. In this systematic review, we aimed to identify and summarize published advances in the refinement [...] Read more.

Refining experiments and housing conditions so as to cause the minimum possible pain and distress is one of the three principles (3Rs) on which Directive 2010/63/EU is based. In this systematic review, we aimed to identify and summarize published advances in the refinement protocols made by European Union-based research groups from 2011 to 2021, and to determine whether or not said research was supported by European or national grants. We included 48 articles, the majority of which were related to improvements in experimental procedures (37/77.1%) for mice (26/54.2%) and were written by research groups belonging to universities (36/57.1%) and from the United Kingdom (21/33.9%). More than two thirds (35/72.9%) of the studies received financial support, 26 (mostly British) at a national level and 8 at a European level. Our results indicated a clear willingness among the scientific community to improve the welfare of laboratory animals, as although funding was not always available or was not specifically granted for this purpose, studies were published nonetheless. However, in addition to institutional support based on legislation, more financial support is needed. We believe that more progress would have been made in refinement during these years if there had been more specific financial support available at both the national and European Union levels since our data suggest that countries investing in refinement have the greatest productivity in successfully publishing refinements. Full article

(This article belongs to the Special Issue Implementing the 3Rs in Laboratory Animal Research—From Theory to Practice)

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