Animal Models In Biomedical Research Research Articles

Response acquisition by zebrafish (Danio rerio) with delayed reinforcement.

Zebrafish (Danio rerio) is a common vertebrate animal model in biomedical research and is a promising species for studying how genes interact with environmental factors in determining behavior. The present study investigated how reinforcement parameters affect zebrafish behavior by assessing response acquisition with delayed reinforcement, which has been studied with other species (e.g., rats, pigeons, humans, etc.) but not with zebrafish. Twenty-four experimentally naïve subjects were exposed to a tandem fixed-ratio 1 differential-reinforcement-of-other-behavior x-s schedule of reinforcement, where x varied across subjects. There were six different delay-to-reinforcement durations and sets of four fish were assigned to each delay duration. All of the fish assigned to a 0-, 0.5-, or 1-s delay acquired responding. Two fish acquired responding with a 3-s delay and one fish appeared to have acquired it with a 6-s delay although the latter result was less clear. None acquired responding with a 12-s delay. These results suggest that zebrafish behavior is sensitive to delays to reinforcement and the time frame over which reinforcement is effective may be limited approximately to 6 s. This time frame is shorter than that found with other species. Practical and theoretical implications of the present finding are discussed.

Development and characterization of 29 SNP markers for the Tibetan macaque (Macaca thibetana)

Macaca thibetana is an endemic species to China that is listed as near threatened in the IUCN Red Data Book. The species has previously been used in animal models for biomedical research. Due to anthropogenic impacts, the wild populations of M. thibetana have dramatically declined in recent years, which has resulted in low genetic diversity. Insufficient molecular markers have hindered the effective conservation and management of this species. In the present study, ten contig sequences containing 29 novel polymorphic SNPs were discovered using 40 samples as templates for PCR validation. The minor allele frequency ranged from 0.100 to 0.488, and the observed and expected heterozygosities ranged from 0 to 0.600 and 0.182 to 0.506, respectively. The FIS ranged from − 0.203 to 1.000. Among these SNPs, four loci showed significant departures from the Hardy–Weinberg equilibrium (p < 0.05). The novel polymorphic SNPs will help elucidate the population structure and conservation genetics of this precious species.

An explorative study on the effect of provision of preferred and non-preferred enrichment on behavioural and physiological parameters in laboratory ferrets (Mustela putorius furo)

Environmental enrichment is often advocated to refine animal studies. Despite the increasing use of ferrets as an animal model in biomedical research, the knowledge on effects of the provision of enrichment on these animals is limited. Additionally, it is unknown whether varying types of enrichment (i.e. preferred and non-preferred) have a different effect. Therefore, to explore the behavioural and physiological effects of providing (differently valued) enrichment to ferrets, three groups of six female ferrets were housed in standard conditions (with bedding, a flexible bucket, a food bowl and a water nipple), with additional non-preferred enrichment (with two ferret balls, a golf ball and an extra food bowl) and with additional preferred enrichment (with two hammocks, a foraging ball and a water bowl) for eight weeks. At the beginning and end of this period, behavioural (i.e. time spent on food and water intake, elimination, maintenance, inactivity, enrichment interaction, exploration, play, and agonistic behaviour) and physiological (i.e. bodyweight and Neutrophil/Lymphocyte ratio [N/L ratio]) parameters were recorded and compared. Results showed that agonistic behaviour increased in the ferrets housed in standard conditions, which was not observed in ferrets that were provided with preferred or non-preferred enrichment. In addition, the ferrets housed with preferred enrichment showed an increase in social play behaviour and a decrease in rearing behaviour (as part of the exploratory behaviours) which were not observed in the ferrets housed in standard conditions or with non-preferred enrichment. Moreover, the ferrets housed with preferred enrichment showed a clear preference for being inactive in the hammock and drinking from the water bowl. As there was only one cage per condition, the results of this explorative study should be considered preliminary and tentative. Nevertheless, the results are a first indication that providing laboratory ferrets with preferred enrichment has positive effects on their behaviour that are not observed in ferrets provided with non-preferred enrichment or housed in standard conditions. Therefore, we recommend to house laboratory ferrets with a hammock, foraging ball and water bowl as these enrichments might help to refine studies using ferrets.

MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice.

The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

Scrotal ablation and orchiectomy in the domestic laboratory goat (Capra hircus)

Domestic goats (Capra hircus) have been used as animal models in biomedical research, and for numerous years for agricultural research and models for human diseases. This column describes an improved surgical technique, for castration of male goats, similar to companion animal techniques for use in the laboratory setting. The technique discussed supports a more in-depth perioperative protocol for performing scrotal ablation and orchiectomy in the male goat.

Proinflammatory and Immunoregulatory Functions of Interleukin 6 as Identified by Reverse Genetics

Reverse genetics approach, involving genome editing, makes it possible not only to establish the nonredundant and unique functions of genes and their products, but also to construct animal models for biomedical research. Interleukin 6 (IL-6) is an important immunoregulatory and proinflammatory cytokine that differs from many related proteins in having a rather complicated signal transduction scheme. Apart from the multiple functions of IL-6, the most relevant biological problem of recent years was establishing what cells produce IL-6, in what form IL-6 is produced, what cells are recipients of the IL-6 signal, and what are the downstream events and physiological consequences of the IL-6 signaling cascade. Because IL-6 is involved in the pathogenesis of many diseases and is a drug target, understanding the mechanisms of its normal and pathogenic effects is important for the clinics. The review summarizes the recent data available in the field.

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies.

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies.

Differences in bone structure and unloading-induced bone loss between C57BL/6N and C57BL/6J mice.

The C57BL/6 mouse, the most frequently utilized animal model in biomedical research, is in use as several substrains, all of which differ by a small array of genomic differences. Two of these substrains, C57BL/6J (B6J) and C57BL/6N (B6N), are commonly used but it is unclear how phenotypically similar or different they are. Here, we tested whether adolescent B6N mice have a bone phenotype and respond to the loss of weightbearing differently than B6J. At 9 weeks of age, normally ambulating B6N had lower trabecular bone volume fraction but greater bone formation rates and osteoblast surfaces than corresponding B6J. At 11 weeks of age, differences in trabecular indices persisted between the substrains but differences in cellular activity had ceased. Cortical bone indices were largely similar between the two substrains. Hindlimb unloading (HLU) induced similar degeneration of trabecular architecture and cellular activity in both substrains when comparing 11-week-old HLU mice to 11-week-old controls. However, unloaded B6N mice had smaller cortices than B6J. When comparing HLU to 9 weeks baseline control mice, deterioration in trabecular separation, osteoblast indices, and endocortical variables was significantly greater in B6N than B6J. These data indicate specific developmental differences in bone formation and morphology between B6N and B6J mice, giving rise to a differential response to mechanical unloading that may be modulated, in part, by the genes Herc2, Myo18b, and Acan. Our results emphasize that these substrains cannot be used interchangeably at least for investigations in which the phenotypic makeup and its response to extraneous stimuli are of interest.

Operant models of relapse in zebrafish (Danio rerio): Resurgence, renewal, and reinstatement

Zebrafish are a widely used animal model in biomedical research, as an alternative to mammals, for having features such as a fully sequenced genome, high fecundity, and low-cost maintenance, but behavioral research with these fish remains scarce. The present study investigated whether zebrafish could be a new animal model for studies on the relapse of behavior (e.g., addiction and overeating) after the behavior has been extinguished. Specifically, we examined whether zebrafish would show three different types of relapse commonly studied with other species: resurgence, renewal, and reinstatement. For resurgence, a target response (i.e., approaching a sensor) was established by presenting a reinforcer (i.e., shrimp eggs) contingent upon the response in Phase 1; the target response was extinguished while introducing reinforcement for an alternative response in Phase 2; neither response produced the reinforcer in Phase 3. For renewal, a target response was established under Context A in Phase 1 and was extinguished under Context B in Phase 2; the fish were placed back in Context A in Phase 3, where extinction remained in effect. For reinstatement, a target response was established in Phase 1 and was extinguished in Phase 2; the reinforcer was presented independently of responding in Phase 3. Each type of relapse occurred in Phase 3. These results replicate and extend previous findings on relapse to a new species and suggest that zebrafish can be a useful animal model for studying the interactions of biological and environmental factors that lead to relapse.

A comprehensive profiling of T- and B-lymphocyte receptor repertoires from a Chinese-origin rhesus macaque by high-throughput sequencing

Due to the close genetic background, high similarity of physiology, and susceptibility to infectious and metabolic diseases with humans, rhesus macaques have been widely used as an important animal model in biomedical research, especially in the study of vaccine development and human immune-related diseases. In recent years, high-throughput sequencing based immune repertoire sequencing (IR-SEQ) has become a powerful tool to study the dynamic adaptive immune responses. Several previous studies had analyzed the responses of B cells to HIV-1 trimer vaccine or T cell repertoire of rhesus macaques using this technique, however, there are little studies that had performed a comprehensive analysis of immune repertoire of rhesus macaques, including T and B lymphocytes. Here, we did a comprehensive analysis of the T and B cells receptor repertoires of a Chinese rhesus macaque based on the 5’—RACE and IR-SEQ. The detailed analysis includes the distribution of CDR3 length, the composition of amino acids and nucleotides of CDR3, V, J and V-J combination usage, the insertion and deletion length distribution and somatic hypermutation rates of the framework region 3 (FR3). In addition, we found that several positions of FR3 region have high mutation frequencies, which may indicate the existence of new genes/alleles that have not been discovered and/or collected into IMGT reference database. We believe that a comprehensive profiling of immune repertoire of rhesus macaque will facilitate the human immune-related diseases studies.

Reversal learning and resurgence of operant behavior in zebrafish (Danio rerio)

Zebrafish are used extensively as vertebrate animal models in biomedical research for having such features as a fully sequenced genome and transparent embryo. Yet, operant-conditioning studies with this species are scarce. The present study investigated reversal learning and resurgence of operant behavior in zebrafish. A target response (approaching a sensor) was reinforced in Phase 1. In Phase 2, the target response was extinguished while reinforcing an alternative response (approaching a different sensor). In Phase 3, extinction was in effect for the target and alternative responses. Reversal learning was demonstrated when responding tracked contingency changes between Phases 1 and 2. Moreover, resurgence occurred in 10 of 13 fish in Phase 3: Target response rates increased transiently and exceeded rates of an unreinforced control response. The present study provides the first evidence with zebrafish supporting reversal learning between discrete operant responses and a laboratory model of relapse. These findings open the possibility to assessing genetic influences of operant behavior generally and in models of relapse (e.g., resurgence, renewal, reinstatement).

Characteristics and Unmet Clinical Needs Related to Hepatocellular Carcinoma

AbstractAdvances in biomedical research require animal models that accurately recapitulate human disease. Without such models, progress against human diseases such as cancer is significantly hindered. Here, we present the current landscape on available and emerging hepatocellular carcinoma (HCC) animal models. HCC is the second leading cause of cancer death worldwide, with an annual death toll exceeding 745,000. Stunningly, only 15% of HCC patients are candidates for curative therapy, leading 85% of patients to seek palliative therapeutic options. The VX2 rabbit model is considered the most relevant and widely used HCC model; however, more reliable HCC models are critically needed. In general, animal models for biomedical research should (1) mimic the human disease on a molecular basis, (2) derive from a relevant cell line that lends itself to in vitro study, (3) be reliable and predictable, (4) manifest survival differences, (5) allow for accurate treatment assessment, (6) be readily imaged, and (7) occur in similar background settings as the human disease. Over the past decades, numerous small animal models have been utilized for HCC studies; however, the development of new large animal models as qualified alternatives to murine models represents a key technology to advance research into human clinical trials.

210 GENERATION OF APOBEC3CH AND APOBEC3H DOUBLE-KNOCKOUT CATS BY SITE-SPECIFIC GENE TARGETING

The domestic cat (Felis catus) is a useful animal model for biomedical research because it shares many devastating diseases with humans. For example, feline immunodeficiency virus-1 is structurally and functionally similar to human immunodeficiency virus-1 (HIV-1). In vitro, the replication of HIV-1 in cat cells is restricted by the feline APOBEC3H (fA3H) and APOBEC3CH (fA3CH). Accordingly, we hypothesised that cats could be used to study HIV-1 infection in humans if fA3H and fA3CH were knocked out. However, due to limited availability of genomic editing tools in the cat, genetic modification has not been widely reported in this species. Here, we show that the fA3H/fA3CH locus could be knocked out in cat using the CRISPR/Cas9 system. Taking advantage of the fact that the fA3H and fA3CH genes share their last four exons (exons 2–5 for fA3H and exons 5–8 for fA3CH), we PCR-amplified and analysed the partial sequence of fA3H and fA3CH and designed a single guide (sg) RNA targeting exon 3 of fA3H (exon 6 of fA3CH) to achieve double knockout of these 2 genes. After transfecting cells with a sgRNA/Cas9 expression DNA vector or co-transfecting the sgRNA along with Cas9 mRNA, which were prepared by in vitro transcription into cat embryonic fibroblast cells, in vitro gene-targeting analysis revealed that the CRISPR/Cas9 system could introduce indels with high efficiency: 23 and 41% of cat fibroblast cells were targeted in the fA3H/fA3CH locus when Cas9 was introduced in the DNA and mRNA forms, respectively. We chose to perform cytoplasmic microinjection injections 6 h post-IVF with 10 pL of injection solution containing 50 ng µL−1 for sgRNA and 100 ng µL−1 for Cas9 mRNA at a volume ratio of 1:1. We achieved a 95.5% embryo survival rate and a 22.9% blastocyst formation rate. High gene-targeting efficiency (62.5%, 15/24) from single blastocyst was achieved in targeting the fA3H/fA3CH locus by PCR-RFLP assay. We transferred 45 injected embryos to the uterine tubes of 4 pseudo-pregnant queens within 2 h of injection. Two of the recipients were determined to be pregnant by ultrasound on Day 30 after embryo transfer, and gave birth to 6 live kittens on Day 65. Genotyping analysis revealed that 2 kittens were successfully targeted in the fA3H/fA3CH locus: kitten 1 was biallelically targeted, whereas kitten 5 was monoallelically targeted. Sanger sequencing of the PCR products subcloned into TA cloning vectors showed that kitten 1 carried the same 1-nucleotide deletion on both alleles, and that kitten 5 also carried the same 1-nucleotide deletion monoallelically. Therefore, we conclude that the fA3H/fA3CH locus in the 2 kittens is specifically targeted by the CRISPR/Cas9 system. To our knowledge, this is the first report of successful site-specific genetic modification in the domestic cat achieved using the CRISPR/Cas9 system and the production of live fA3H/fA3CH double-knockout kittens to study the HIV-1 infected diseases. Supported by the BioGreen 21 (PJ01107703, PJ01107704), Felix Pets, and BK21+ program, Republic of Korea.

Complex and interacting influences of the autonomic nervous system on cardiac electrophysiology in conscious mice.

Mice may now be the preferred animal model for biomedical research due to its anatomical, physiological, and genetic similarity to humans. However, little is known about accentuated antagonism of chronotropic and dromotropic properties in conscious mice. Accordingly, we describe the complex and interacting influence of the autonomic nervous system on cardiac electrophysiology in conscious mice. Specifically, we report the effects of single and combined cardiac autonomic blockade on measurements of pulse interval (heart rate), atrio-ventricular interval, sinus node recovery time (SNRT), SNRT corrected for spontaneous sinus cycle, and Wenckebach cycle length in conscious mice free of the confounding influences of anesthetics and surgical trauma. Autonomic influences were quantified as the change in parameter induced by its selective blocker (Sympathetic or Parasympathetic Effect) or as the difference between the intrinsic value and the value after a selective blocker (Sympathetic or Parasympathetic Tonus). Sympatho-Vagal Balance (SVB) was assessed as the ratio of control interval to intrinsic interval. SVB suggests slight parasympathetic dominance in the control of cardiac electrophysiology intervals. Furthermore, results documents a complex interaction between the sympathetic and parasympathetic divisions of the autonomic nervous system in the control of cardiac electrophysiology parameters. Specifically, the parasympathetic effect was greater than the parasympathetic tonus in the control of cardiac electrophysiology parameters. In contrast, the sympathetic effect was smaller than the sympathetic tonus in the control of cardiac electrophysiology parameters. Results have important implications because actions of pharmacological agents that alter the autonomic control of cardiac electrophysiology are transformed by these interacting mechanisms.

Gene conversion of the major histocompatibility complex class I Caja-G in common marmosets (Callithrix jacchus).

SummaryCurrently, the amount of sequenced and classified MHC class I genes of the common marmoset is limited, in spite of the wide use of this species as an animal model for biomedical research. In this study, 480 clones of MHC class I G locus (Caja‐G) cDNA sequences were obtained from 21 common marmosets. Up to 10 different alleles were detected in each common marmoset, leading to the assumption that the Caja‐G loci duplicated in the marmoset genome. In the investigated population, four alleles occurred more often, giving evidence for higher immunological advantage of these alleles. In contrast to the human non‐classical MHC class I genes, Caja‐G shows high rates of polymorphism at the relevant peptide‐binding sites, despite its phylogenetic relationship to the non‐classical HLA‐G. Our results provide information for better understanding of the immunological properties of the common marmoset and confirm the theory of a gene conversion of the Caja‐G due to its detected plasticity and the absence of any known HLA‐A equivalent.

Hybrid multispectral optoacoustic and ultrasound tomography for morphological and physiological brain imaging.

Expanding usage of small animal models in biomedical research necessitates development of technologies for structural, functional, or molecular imaging that can be readily integrated in the biological laboratory. Herein, we consider dual multispectral optoacoustic (OA) and ultrasound tomography based on curved ultrasound detector arrays and describe the performance achieved for hybrid morphological and physiological brain imaging of mice in vivo. We showcase coregistered hemodynamic parameters resolved by OA tomography under baseline conditions and during alterations of blood oxygen saturation. As an internal reference, we provide imaging of abdominal organs. We illustrate the performance advantages of hybrid curved detector ultrasound and OA tomography and discuss immediate and long-term implications of our findings in the context of animal and human studies.

Comparative genomics of the human, macaque and mouse major histocompatibility complex.

The MHC is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. The MHC locus was first discovered in the mouse and for the past 50 years it has been studied most intensively in both mice and humans. However, in recent years the macaque species have emerged as some of the more important and advanced experimental animal models for biomedical research into MHC with important human immunodeficiency virus/simian immunodeficiency virus and transplantation studies undertaken in association with precise MHC genotyping and haplotyping methods using Sanger sequencing and next-generation sequencing. Here, in this special issue on 'Macaque Immunology' we provide a short review of the genomic similarities and differences among the human, macaque and mouse MHC class I and class II regions, with an emphasis on the association of the macaque class I region with MHC polymorphism, haplotype structure and function.

Cynomolgus macaque (Macaca fascicularis) immunoglobulin heavy chain locus description.

Cynomolgus macaques (Macaca fascicularis) have become an important animal model for biomedical research. In particular, it is the animal model of choice for the development of vaccine candidates associated with emerging dangerous pathogens. Despite their increasing importance as animal models, the cynomolgus macaque genome is not fully characterized, hindering molecular studies for this model. More importantly, the lack of knowledge about the immunoglobulin (IG) locus organization directly impacts the analysis of the humoral response in cynomolgus macaques. Recent advances in next generation sequencing (NGS) technologies to analyze IG repertoires open the opportunity to deeply characterize the humoral immune response. However, the IG locus organization for the animal is required to completely dissect IG repertoires. Here, we describe the localization and organization of the rearranging IG heavy (IGH) genes on chromosome 7 of the cynomolgus macaque draft genome. Our annotation comprises 108 functional genes which include 63 variable (IGHV), 38 diversity (IGHD), and 7 joining (IGHJ) genes. For validation, we provide RNA transcript data for most of the IGHV genes and all of the annotated IGHJ genes, as well as proteomic data to validate IGH constant genes. The description and annotation of the rearranging IGH genes for the cynomolgus macaques will significantly facilitate scientific research. This is particularly relevant to dissect the immune response during vaccination or infection with dangerous pathogens such as Ebola, Marburg and other emerging pathogens where non-human primate models play a significant role for countermeasure development.

High-Speed Video-Oculography for Measuring Three-Dimensional Rotation Vectors of Eye Movements in Mice.

BackgroundThe mouse is the most commonly used animal model in biomedical research because of recent advances in molecular genetic techniques. Studies related to eye movement in mice are common in fields such as ophthalmology relating to vision, neuro-otology relating to the vestibulo-ocular reflex (VOR), neurology relating to the cerebellum’s role in movement, and psychology relating to attention. Recording eye movements in mice, however, is technically difficult.MethodsWe developed a new algorithm for analyzing the three-dimensional (3D) rotation vector of eye movement in mice using high-speed video-oculography (VOG). The algorithm made it possible to analyze the gain and phase of VOR using the eye’s angular velocity around the axis of eye rotation.ResultsWhen mice were rotated at 0.5 Hz and 2.5 Hz around the earth’s vertical axis with their heads in a 30° nose-down position, the vertical components of their left eye movements were in phase with the horizontal components. The VOR gain was 0.42 at 0.5 Hz and 0.74 at 2.5 Hz, and the phase lead of the eye movement against the turntable was 16.1° at 0.5 Hz and 4.88° at 2.5 Hz.ConclusionsTo the best of our knowledge, this is the first report of this algorithm being used to calculate a 3D rotation vector of eye movement in mice using high-speed VOG. We developed a technique for analyzing the 3D rotation vector of eye movements in mice with a high-speed infrared CCD camera. We concluded that the technique is suitable for analyzing eye movements in mice. We also include a C++ source code that can calculate the 3D rotation vectors of the eye position from two-dimensional coordinates of the pupil and the iris freckle in the image to this article.

Endocrinopathy and Aging in Ferrets

Ferrets have become more popular as household pets and as animal models in biomedical research in the past 2 decades. The average life span of ferrets is about 5-11 years with onset of geriatric diseases between 3-4 years including endocrinopathies, neoplasia, gastrointestinal diseases, cardiomyopathy, splenomegaly, renal diseases, dental diseases, and cataract. Endocrinopathies are the most common noninfectious disease affecting middle-aged and older ferrets. Spontaneous neoplasms affecting the endocrine system of ferrets appear to be increasing in prevalence with a preponderance toward proliferative lesions in the adrenal cortex and pancreatic islet cells. Diet, gonadectomy, and genetics may predispose ferrets to an increased incidence of these endocrinopathies. These functional proliferative lesions cause hypersecretion of hormones that alter the physiology and metabolism of the affected ferrets resulting in a wide range of clinical manifestations. However, there is an apparent dearth of information available in the literature about the causal relationship between aging and neoplasia in ferrets. This review provides a comprehensive overview of the anatomy and physiology of endocrine organs, disease incidence, age at diagnosis, clinical signs, pathology, and molecular markers available for diagnosis of various endocrine disorders in ferrets.