Animal Welfare Considerations Research Articles

Views of American animal and dairy science students on the future of dairy farms and public expectations for dairy cattle care: A focus group study

Students completing advanced degrees in dairy or animal science may go on to have a major impact on the food animal agriculture industries. The aim of this study was to better understand student views of the future of dairying, including changes in practices affecting animal care on farms as well as perceived public perceptions. We conducted 6 focus group sessions with undergraduate students enrolled in the 2019 US Dairy Education and Training Consortium held in Clovis, New Mexico, and used explorative key word analysis of written notes and thematic analysis of the semi-structured discussions. Some "must-haves" of future animal care on dairy farms included increased use of technology, group housing of calves, and adequate facilities, including enrichment. Students also discussed their views of public expectations regarding animal care on dairy farms, and measures that they felt must be put into place to address these expectations in the coming years. Although the influence of the public was highlighted by the students, they were not always certain what specific values the public holds and doubted the feasibility and practicality of some expectations, such as providing pasture access or keeping the calf and cow together. They further demonstrated uncertainty about how best to align the directions of the industry with public expectations. Although they felt that public education could be used to demonstrate the legitimacy of dairy practices, they also believed that the industry should strive to find compromises and work toward meeting public expectations. Deciding what animal welfare considerations (e.g., naturalness, affective states, or animal health) were most relevant was a challenge for the students, perhaps reflecting diverging messages received during their own education.

Therapy of bullous pemphigoid in a Warmblood gelding

A 15-year-old Warmblood gelding was presented with multiple large, ulcerative, and crusty dermal lesions that had been existing for 4 years. Histopathology of a skin biopsy revealed cleft formation at the dermal-epidermal junction beneath the basal cells and above the basement membrane leading to the diagnosis of bullous pemphigoid. Immunosuppressive therapy with dexamethasone and azathioprine was initiated and after 14 weeks full remission of the ulcers was achieved. Scar tissue formation was evident in the areas of the formerly affected lesions. Following medication tapering over a period of 5 months, long-term therapy was continued with a maintenance dose of 0.5 mg/kg azathioprine daily. The ulcerative lesions recurred after 63 weeks of disease stabilization. Additionally, adverse drug reactions (acute laminitis and increased susceptibility to infections) were evident and the gelding was euthanized due to animal welfare considerations.

ImmuneDEX: a strategy for the genetic improvement of immune competence in Australian Angus cattle.

In animal breeding and genetics, the ability to cope with disease, here defined as immune competence (IC), with minimal detriment to growth and fertility is a desired objective which addresses both animal production and welfare considerations. However, defining and objectively measuring IC phenotypes using testing methods which are practical to apply on-farm has been challenging. Based on previously described protocols, we measured both cell-mediated immune response (Cell-IR) and antibody-mediated immune response (Ab-IR) and combined these measures to determine an animal's IC. Using a population of 2,853 Australian Angus steers and heifers, we compared 2 alternative methods to combine both metrics into a single phenotype to be used as a tool for the genetic improvement of IC. The first method, named ZMEAN, is obtained by taking the average of the individual metrics after subjecting each to a Z-score standardization. The second, ImmuneDEX (IDEX), is a weighted average that considers the correlation between Cell-IR and Ab-IR, as well as the difference in ranking of individuals by each metric, and uses these as weights in the averaging. Both simulation and real data were used to understand the behavior of ZMEAN and IDEX. To further ascertain the relationship between IDEX and other traits of economic importance, we evaluated a range of traits related to growth, feedlot performance, and carcass characteristics. We report estimates of heritability of 0.31 ± 0.06 for Cell-IR, 0.42 ± 0.06 for Ab-IR, 0.42 ± 0.06 for ZMEAN and 0.370 ± 0.06 for IDEX, as well as a unity genetic correlation (rg) between ZMEAN and IDEX. While a moderately positive rg was estimated between Cell-IR and Ab-IR (rg = 0.33 ± 0.12), strongly positive estimates were obtained between IDEX and Cell-IR (rg = 0.80 ± 0.05) and between IDEX and Ab-IR (rg = 0.85 ± 0.04). We obtained a moderately negative rg between IC traits and growth including an rg = -0.38 ± 0.14 between IDEX and weaning weight, and negligible with carcass fat measurements, including an rg = -0.03 ± 0.12 between IDEX and marbling. Given that breeding with a sole focus on production might inadvertently increase susceptibility to disease and associated antibiotic use, our analyses suggest that ImmuneDEX will provide a basis to breed animals that are both highly productive and with an enhanced ability to resist disease.

Identification of Individual Zebrafish (Danio rerio): A Refined Protocol for VIE Tagging Whilst Considering Animal Welfare and the Principles of the 3Rs.

Simple SummaryIn aquatic ecology research studies commonly employ a tagging technique named visible implant elastomer (VIE). Despite existing widespread guidance on the use of this marking technique on fish, there is still a lack of information regarding efficiency in small fishes, as well as its impact on fish welfare. The current paper highlights important animal welfare issues and introduces a newly improved VIE tagging protocol, presenting information on individual survival rate, tag retention, and use of different elastomer colours, quantified in different populations and age groups of zebrafish. Specifically, we compare a previously used tagging method with a newly improved protocol which places particular emphasis to the Three Rs, helping to refine this scientific procedure. The shared detailed protocol and information will be beneficial to the zebrafish research community and beyond.In aquatic ecology, studies have commonly employed a tagging technique known as visible implant elastomer (VIE). This method has not been widely adopted by the zebrafish research community and also lacks refinement with regard to animal welfare. The current paper introduces a new VIE tagging protocol, with the aim of improving existing tagging techniques by placing particular emphasis on the Three Rs. To improve animal welfare and fish survival, we added the use of an analgesic compound (lidocaine) through the marking procedure, followed by after-treatment with antiseptics (melaleuca, aloe vera, and PVP-I as active ingredients) to improve tissue regeneration and healing. The newly improved protocol has been quantitatively evaluated on different populations and age groups of zebrafish. This study will be useful to the scientific zebrafish community and to the wider field including biologist and aquarists, especially in consideration of animal welfare, where tagging techniques are considered as a potential noxious stimulus for fish.

Organic dairy producer experiences and decisions related to disease prevention and treatment

Organic dairy producers have distinct perspectives, approaches, challenges, and experiences when managing herd health, but little research has been conducted to develop a deeper understanding of producer decision-making related to disease-prevention and treatment strategies. We used 23 semi-structured interviews with organic dairy producers to better understand their perceived herd health challenges and overall disease-prevention strategies; decision-making and attitudes related to vaccination; and approaches to the treatment of infectious diseases using alternative or conventional antimicrobial therapies. Organic dairy producers reported improvements in overall animal health after they transitioned from a conventional to an organic system. Outdoor access, a balanced forage-based diet, and animal and environmental cleanliness were reported as the predominant disease-prevention measures. Over half of producers vaccinated their animals, and almost all preferred to use vaccines sparingly. Among producers who did not use vaccines, reasons cited were low perceived risk for infectious disease and minor vaccine safety concerns, yet many were not opposed to using vaccines in the future. Non-antimicrobial therapies were viewed as alternative effective treatment strategies for most common infectious diseases. Producers used antimicrobials very rarely and generally reserved them for severe cases when alternative treatments failed. Because US organic standards prohibit selling milk from cows that have ever been treated with antimicrobials, producers described the decision to use antimicrobials as extremely challenging and involved balancing considerations of economics, convenience, and animal welfare. This research fosters a better understanding of organic dairy producers' disease-prevention and treatment strategies and could be used by veterinarians and other animal health specialists to facilitate services for organic clients.

Animal Welfare Considerations among Dairy Consumers in Rajasthan, India

Animal Welfare Considerations among Dairy Consumers in Rajasthan, India

Limitations of animal epilepsy research models: Can epileptic human tissue provide translational benefit?

Advancement of understanding the etiology and treatment of epilepsy has largely depended on the use of acute and chronic animal models. An alternative approach, which is being increasingly used by a select number of laboratories worldwide, is to perform functional mechanistic studies in brain slices of living human tissue resected during surgery for drug resistant epilepsies. Pharmacoresistant epilepsy is a major clinical problem with a significant proportion of patients not receiving any symptomatic benefit from available anti-epileptic drugs. Animal models of epilepsy have dominated the landscape with regard to research and development, however they have failed to deliver new agents that would provide seizure control in patients with drug refractory epilepsy. Moreover, these models have considerable issues with respect to validity and animal welfare considerations. A compelling alternative is the use of live human epileptic tissue, which recapitulates a number of key features of refractory epilepsy. The use of live epileptic human tissue offers unprece­dented opportunities to understand the mechanisms associated with difficult to treat epilepsy whilst also permitting studies of efficacy of novel agents that are being developed to alleviate epilepsy in drug resistant patients.

Research Relevant Conditions and Pathology in Nonhuman Primates.

Biomedical research involving animal models continues to provide important insights into disease pathogenesis and treatment of diseases that impact human health. In particular, nonhuman primates (NHPs) have been used extensively in translational research due to their phylogenetic proximity to humans and similarities to disease pathogenesis and treatment responses as assessed in clinical trials. Microscopic changes in tissues remain a significant endpoint in studies involving these models. Spontaneous, expected (ie, incidental or background) histopathologic changes are commonly encountered and influenced by species, genetic variations, age, and geographical origin of animals, including exposure to infectious or parasitic agents. Often, the background findings confound study-related changes, because numbers of NHPs used in research are limited by animal welfare and other considerations. Moreover, background findings in NHPs can be exacerbated by experimental conditions such as treatment with xenobiotics (eg, infectious morphological changes related to immunosuppressive therapy). This review and summary of research-relevant conditions and pathology in rhesus and cynomolgus macaques, baboons, African green monkeys, common marmosets, tamarins, and squirrel and owl monkeys aims to improve the interpretation and validity of NHP studies.

Hormone data collection in support of endocrine disruption (ED) assessment for aquatic vertebrates: Pragmatic and animal welfare considerations

Hormone data collection in support of endocrine disruption (ED) assessment for aquatic vertebrates: Pragmatic and animal welfare considerations

Why Are Octopuses Going to Be the ‘Poster Child’ for Invertebrate Welfare?

ABSTRACT Animal welfare consideration and actions are generally addressed to animals similar to us, predominantly large mammals. Invertebrates are neglected partly because they are unknown, though new exploration of the oceans has helped with this. Also, we know little about their ecology and welfare. This is gradually changing, and the octopuses are likely to be the first beneficiaries. Scientists are finding that cephalopods are far more intelligent than we thought, with the Cambridge Declaration of Consciousness suggesting they might possess this quality of mind. Partly as a result, the European Union has described and demanded good care for cephalopods such as the octopus in captivity. Public opinion has been swayed to approval by anecdotes of octopuses doing unusual actions, and by several recent books pointing out interesting and intelligent behavior of cephalopods. Aquariums have begun to feature octopuses for them. With this progress, welfare of invertebrate animals has begun to matter. While the octopuses will be the first animal group to benefit, they may pave the way for us to see that different does not mean unworthy of regard and welfare consideration.

Preferences for Certified Beef with Animal Welfare and Other Credence Attributes in Australia

Concerns over livestock production practices have resulted in increased consumer preferences for certified products. Australian beef buyers’ survey data revealed the preferences of consumers who would buy differentiated beef based on animal welfare, safety, health, or environmental-friendly considerations. Female respondents are more likely to buy certified animal welfare products. Buyers with children, and those who value branded beef, are more likely to buy products differentiated by a bundle of credence attributes. Given that Australian beef consumers eat similar amounts, there are opportunities for differentiating beef products according to credence attributes and offering them in a range of retailers.

Airflow Characteristics Downwind a Naturally Ventilated Pig Building with a Roofed Outdoor Exercise Yard and Implications on Pollutant Distribution

The application of naturally ventilated pig buildings (NVPBs) with outdoor exercise yards is on the rise mainly due to animal welfare considerations, while the issue of emissions from the buildings to the surrounding environment is important. Since air pollutants are mainly transported by airflow, the knowledge on the airflow characteristics downwind the building is required. The objective of this research was to investigate airflow properties downwind of a NVPB with a roofed outdoor exercise yard for roof slopes of 5°, 15°, and 25°. Air velocities downwind a 1:50 scaled NVPB model were measured using a Laser Doppler Anemometer in a large boundary layer wind tunnel. A region with reduced mean air velocities was found along the downwind side of the building with a distance up to 0.5 m (i.e., 3.8 times building height), in which the emission concentration might be high. Additional air pollutant treatment technologies applied in this region might contribute to emission mitigation effectively. Furthermore, a wake zone with air recirculation was observed in this area. A smaller roof slope (i.e., 5° slope) resulted in a higher and shorter wake zone and thus a shorter air pollutant dispersion distance.

Effects of Drinking Water Temperature and Flow Rate during Cold Season on Growth Performance, Nutrient Digestibility and Cecum Microflora of Weaned Piglets.

Simple SummaryWater is an essential nutrient pigs need to sustain life and ensure growth. Determining the appropriate drinking water supply parameters during cold weather are critical for the welfare and growth of pigs, especially vulnerable weaned piglets. This study explored different combinations of drinking water temperature (DWT) and flow rate (DWF) for weaned piglets during winter. It measured their growth performance, nutrient digestibility and cecum microbial diversity, aiming to figure out the optimal water supplying conditions for weaned piglets. The results indicated that a combination of DWT of 30 °C and DWF of 300 mL/min decreased diarrhea occurrence. Furthermore, this increased growth performance and nutrient digestibility, accompanied by improvement of the dominant cecum microflora, mainly manifested in a reduced abundance of Proteobacteria and increased abundance of Bacteroidetes. This study enriches our understanding of the connection between water supply, growth performance and cecum microbiota on weaned piglets during the cold season.Although water is one of the most important nutrients and is essential for various physiological processes within the body, it does not receive adequate consideration when ensuring optimal nutrition and growth performance in piglets. This study was conducted to investigate the effects of drinking water temperature (DWT) and flow rate (DWF) on growth performance, nutrient digestibility and cecum microflora in weaned piglets during cold weather. Sixty-four piglets with an average body weight of 8.60 ± 0.5 kg were allotted into four groups with four replicates in each group and four pigs in each replicate. The DWT and DWF were set for each group as follows: (1) 13 °C + 300 mL/min, (2) 13 °C + 700 mL/min, (3) 30 °C + 300 mL/min and (4) 30 °C + 700 mL/min, respectively. All groups were fed the same diet during the 28 d trial. The body weight at day 0 and day 29, as well as daily feed intake, were recorded. Diarrhea severity was assessed every day. Fresh fecal samples were collected for four consecutive days at the end of the experiment for the digestibility test. Cecum content was collected after sacrifice for microbial composition analysis. The results indicated that: (1) DWT at 30 °C promoted the average daily gain (ADG) of weaned piglets considerably (p = 0.043) and decreased feed to weight ratio when compared with DWT at 13 °C (p = 0.045). DWF had no substantial effect on the growth performance of piglets (p > 0.05). (2) The 30 °C DWT groups had higher apparent digestibility of crude protein, crude fat and energy than the 13 °C DWT groups (p < 0.05), while DWF had no significant effect on the apparent digestibility of nutrients (p > 0.05). (3) DWT at 30 °C increased the Bacteroidetes abundance and decreased the Proteobacteria abundance in cecum digesta. The change in these two factors may be related to a decrease in diarrhea and the improvement of growth performance. Different DWF had no substantial effect on the cecum microbial structure. To sum up, providing a DWT of 30 °C to weaned piglets in cold weather reduced the abundance of harmful bacteria in the cecum and improved the apparent nutrient digestibility, which is beneficial for maintaining a healthy intestinal microenvironment and promoting growth performance. A lower DWF of 300 mL/min had no adverse effect on growth performance. Therefore, a combination of 30 °C + 300 mL/min is recommended for weaned piglets during cold weather for the consideration of animal welfare and production efficiency.

A Review of Enteric Methane Emission Measurement Techniques in Ruminants.

Simple SummaryEnteric methane emissions from ruminants are a major contributor to atmospheric greenhouse gas accumulation in agricultural production systems, which may consequently contribute to climate change. Accurate measurement of methane production in ruminants is vital to develop robust national greenhouse gas emission inventories and to evaluate mitigation strategies of methane emissions. This review summarizes several common methane measurement techniques suitable for ruminant production and discusses the advantages and disadvantages of each method. There is not a perfect technique for all situations. The appropriate technique depends on the objectives and resources available. Sophisticated techniques such as respiration chambers, sulphur hexafluoride tracer and ventilated hood are usually highly accurate but labor and time intensive. Simplified techniques such as GreenFeed, sniffer method, facemask, laser methane detector and portable accumulation chamber may be less accurate but are rapid with a high throughput. In general, an understanding of measurement mechanisms and the advantages and disadvantages with an appreciation of animal behavior and welfare is necessary for all techniques.To identify relationships between animal, dietary and management factors and the resulting methane (CH4) emissions, and to identify potential mitigation strategies for CH4 production, it is vital to develop reliable and accurate CH4 measurement techniques. This review outlines various methods for measuring enteric CH4 emissions from ruminants such as respiration chambers (RC), sulphur hexafluoride (SF6) tracer, GreenFeed, sniffer method, ventilated hood, facemask, laser CH4 detector and portable accumulation chamber. The advantages and disadvantages of these techniques are discussed. In general, RC, SF6 and ventilated hood are capable of 24 h continuous measurements for each individual animal, providing accurate reference methods used for research and inventory purposes. However, they require high labor input, animal training and are time consuming. In contrast, short-term measurement techniques (i.e., GreenFeed, sniffer method, facemask, laser CH4 detector and portable accumulation chamber) contain additional variations in timing and frequency of measurements obtained relative to the 24 h feeding cycle. However, they are suitable for large-scale measurements under commercial conditions due to their simplicity and high throughput. Successful use of these techniques relies on optimal matching between the objectives of the studies and the mechanism of each method with consideration of animal behavior and welfare. This review can provide useful information in selecting suitable techniques for CH4 emission measurement in ruminants.

Consumers’ Evaluation of Stockfree-Organic Agriculture—A Segmentation Approach

Recently, more and more research has been conducted on what sustainable nutrition could look like. Stockfree-organic agriculture is one possible approach but a relatively new and unstudied cultivation method. In addition to organic agriculture, it excludes any animal by-products during the whole cultivation process. Among the consumers of organic food are especially many vegetarians and vegans. To attract this target group, first farms in Europe have started to follow the stockfree-organic agriculture principles. As it is important to know the consumers’ point of view on new developments in agriculture at an early stage of the diffusion process, this study deals with consumers’ evaluation of stockfree-organic agriculture to draw conclusions about a possible market potential. This is especially important for stockfree-organic farmers, as well as for organic farmers who are considering converting their cultivation method, and for retailers who wonder whether it is worthwhile to offer these products. The data was collected in 2019 by means of an online survey. The sample consisted of 500 German respondents. Principal component and cluster analyses were used to identify consumer segments according to their attitudes towards the acceptance, advantages, and disadvantages of stockfree-organic agriculture. Additionally, the different segments were compared with each other according to various attitudes and eating behaviours. Overall, animal welfare considerations and environmental aspects were of particular importance to consumers. Animal usage was clearly rejected by one segment, which contained 24% of the sample. Nearly all vegetarians and all vegans supported stockfree-organic agriculture, whereas heavy meat consumers tended to refuse the support of stockfree-organic agriculture. The supporting group valuing high animal welfare and health concerns was much larger than the current status of this niche segment would suggest. This could be a major challenge for the agricultural sector in the long term, but could also include opportunities for greater sustainability.

Best Practices for Preclinical In Vivo Testing of Cancer Nanomedicines.

Significant advances have been made in the development of nanoparticles for cancer treatment in recent years. Despite promising results in preclinical animal models, cancer nanomedicines often fail in clinical trials. This failure rate could be reduced by defining stringent criteria for testing and quality control during the design and development stages, and by performing carefully planned preclinical studies in relevant animal models. This article discusses best practices for the evaluation of nanomedicines in murine tumor models. First, a recommended set of experiments to perform is introduced, including discussion of the types of data to collect during these studies. This is followed by an outline of various tumor models and their clinical relevance. Next, different routes of nanoparticle administration are overviewed, followed by a summary of important controls to include in in vivo studies of nanomedicine. Finally, animal welfare considerations are discussed, and an overview of the steps involved in achieving US Food and Drug Administration approval after animal studies are completed is provided. Researchers should use this report as a guideline for effective preclinical evaluation of cancer nanomedicine. As the community adopts best practices for in vivo testing, the rate of clinical translation of cancer nanomedicines is likely to improve.

(Q)SAR tools for the prediction of mutagenic properties: Are they ready for application in pesticide regulation?

The assessment of human health risks resulting from the presence of metabolites in groundwater and food residues has become an important element in pesticide authorisation. In this context, the evaluation of mutagenicity is of particular interest and a paradigm shift from exposure-triggered testing to in silico-based screening has been recommended in the European Food Safety Authority (EFSA) Guidance on the establishment of the residue definition for dietary risk assessment. In addition, it is proposed to apply in silico predictions when experimental mutagenicity testing is not possible due to a lack of sufficient quantities of the pesticide metabolite. This, combined with animal welfare and economic considerations, has led to a situation where an increasing number of in silico studies are submitted to regulatory authorities. Whilst there is extensive experience with in silico predictions for mutagenicity in the chemical and pharmaceutical industry, their suitability in pesticide regulation is still insufficiently considered. Therefore, we herein discuss critical issues that need to be resolved to successfully implement (Quantitative) Structure-Activity Relationship ((Q)SAR) as an accepted tool in pesticide regulation. For illustration purposes, the results of a pilot study are included. The presented study highlights a need for further improvement regarding the predictivity and applicability domain of (Q)SAR systems for pesticides and their metabolites, but also raises other questions such as model selection, establishment of acceptance criteria, harmonised approaches to the combination of model outputs into overall conclusions, adequate reporting and data sharing. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Viewpoint: Regulating meat consumption to improve health, the environment and animal welfare

Meat consumption has increased significantly in the last 50 years. This trend raises various health and environmental issues, as well as moral concerns regarding farm animal welfare. In this paper, we discuss the regulation of meat consumption in developed countries. Specifically, we discuss possible justifications for this regulation in terms of environmental, health and animal welfare considerations, as well as the effect of fiscal, informational and behavioral regulatory instruments. Finally, we present a list of challenges that policy makers and food scholars may need to confront in the future.

Shark Fin Regulations in the United States: Animal Welfare, Cultural, and Policy Considerations

Sharks play critical roles in the marine ecosystem, and they face serious threats due to overfishing. Conservation efforts have focused on the consumption of shark fins, especially the “finning” practice that removes the fins of a shark and discards the carcass at sea. This article reviews the shark fin legislation in the United States, including the “finning ban” which outlaws finning practices and the “fin ban” that prohibits the use of shark fins entirely. Our case study specifically focuses on the animal welfare, cultural, and policy debates surrounding these bans. We discuss how and why shark finning is regarded as a cruel practice and whether shark fin bans discriminate against Chinese Americans. At the policy level, there is an ongoing policy debate whether a ban on shark fins in the United States would lead to increased protection of sharks or it would have little effect on the global trade. Due to the lack of detailed information on shark fisheries, the policy discussion is likely to persist. Although this case study focuses only on regulations on shark fins, we would like to emphasize that shark fin industry is not the only threat to sharks. Conservationists also need to consider other issues such as bycatch, habitat destruction, and a wider array of policy tools to protect sharks.

Multiple-endpoint genotoxicity assay for colon carcinogen 1,2-dimethylhydrazine

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R’s) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.