Euthanized Research Articles

ИССЛЕДОВАНИЕ ЭФФЕКТИВНОСТИ РАЛТЕГРАВИРА (“ИСЕНТРЕСС”, MSD, США) ПРИ РЕГЕНЕРАТИВНОЙ АНЕМИИ, АССОЦИИРОВАННОЙ С ВИРУСНОЙ ЛЕЙКЕМИЕЙ КОШЕК

The leukemia virus is widespread in the population of domestic cats and, despite the study of this disease, there is no effective therapy aimed at complete eradication of the virus from the body of an infected animal. One of the most life-threatening conditions associated with the frequent death and euthanasia of infected animals is anemia. This retrospective study is aimed at evaluating the effectiveness of the drug “Isentress” (MSD, USA) with the active substance Raltegravir against regenerative anemia associated with feline viral leukemia. In the group of animals receiving this drug, a decrease in the concentration of the virus in the blood was recorded, as well as an improvement in the hematological parameters of red blood. Hematological indicators and the concentration of the virus showed positive dynamics in one of the cats in the group of animals that did not take Raltegravir, but the connection with the owner of this animal was lost, which does not allow us to draw final conclusions about the nature of these laboratory phenomena. The viral load increased when the drug was discontinued, which characterizes the need for lifelong administration of Raltegravir. There were no side effects requiring withdrawal of the drug. The only registered side effect was lethargy in one of the infected cats, but it was not possible to associate apathy with taking the drug due to the presence of hematological abnormalities associated with severe anemia in the animal at the time of the side effect. Raltegravir has shown efficacy and safety when taken in cats with regenerative anemia associated with feline viral leukemia, however, further studies are required to assess the safety of the drug when taken for a long time in infected cats.

Is it Possible to be Better Off Dead? An Epicurean Analysis of Physician-Assisted Suicide

Epicurus wrote that death cannot be bad for a person who dies, since when someone dies they no longer exist to be the subject of harm. But his conclusion also applies in the converse: Death cannot be good for someone, since after their death they will not exist to be the subject of benefit. This conclusion is troubling when it is brought to bear on the question of physician assisted suicide. If Epicurus is right, as I think he is, then it means that even when someone is dying of a long and painful illness, we cannot say that they would be better off dead. I defend this conclusion by first presenting Epicurus’s argument, and defending it against some of its more important contemporary critics. I then re-examine the conclusion that someone cannot be better off dead by looking at the issue of mercy killing and euthanasia. Drawing on the work of philosophers who examine the ethics of animal euthanasia, I argue that killing someone who is suffering severe agony can be good, but it cannot be good for the person who suffers – it is just good in itself. This, I think, is why we have the intuitions that we do around physician assisted suicide: we are mistaken about the idea that someone can be better off dead, but we can be correct in thinking that sometimes someone’s death is good.

The Effect of Pet Insurance on Presurgical Euthanasia of Dogs With Gastric Dilatation-Volvulus: A Novel Approach to Quantifying Economic Euthanasia in Veterinary Emergency Medicine.

Euthanasia of companion animals in veterinary emergency medicine is a common cause of death. Euthanasia is economic when it is the consequence of the pet owner's inability to afford essential treatment while a viable medical alternative to euthanasia exists. Gastric dilatation-volvulus (GDV) is an acute life-threatening emergency condition of dogs; if left untreated, rapid death is highly likely. Surgical treatment leads to survival of around 80-90% of dogs; however, such treatment is costly. Therefore, pre-surgical euthanasia may be largely economically motivated. Having pet insurance, a financial instrument to reduce the burden of unforeseen veterinary medical costs on pet owners, would be expected to abolish the risk for pre-surgical economic euthanasia. We therefore aimed to determine whether pet insurance attenuates the risk of pre-surgical economic euthanasia in dogs with GDV. Non-referred dogs (n = 260) with GDV and known insurance status seen at 24 emergency clinics over a 2-year period were included. Relevant data (e.g., insurance status, age, comorbidities, outcome) were retrospectively extracted from a pet insurer's claim records (insured animals) or from electronic medical records of participating hospitals (non-insured animals). Forty-one percent of dogs (106 of 260 dogs) did not survive to hospital discharge; 82 (77%) of non-survivors died before surgery, all through euthanasia. The pre-surgical euthanasia rate was 10% in insured and 37% in non-insured dogs (p < 0.001). When adjusted for the effect of age, deposit size, comorbidities, and blood lactate concentration, the absence of insurance increased the odds of pre-surgical euthanasia by a factor of 7.4 (95% CI 2.0 to 37; p = 0.002). Of dogs undergoing surgery, 86% survived to hospital discharge. Overall, 80% of insured animals and 53% of non-insured animals survived to hospital discharge (p < 0.001). Thus, insurance was associated with a marked decrease in risk of pre-surgical euthanasia indicating that the cause of pre-surgical euthanasia of dogs with GDV is predominantly economic in nature. The rate of pre-surgical euthanasia in dogs with GDV may emerge as a suitable marker to quantify economic decision making of pet owners and to measure the impact of financial interventions aimed at mitigating economic duress associated with cost of veterinary emergency care.

The Definition of Nonhuman Animal Euthanasia

Under what conditions does the killing of a nonhuman animal qualify as euthanasia? In this paper, I elaborate an original nonprescriptive definition of nonhuman animal euthanasia which avoids the conceptual confusions surrounding the use of this expression. Such a definition imposes strict limitations on the notion of nonhuman animal euthanasia. On the one hand, the nonhuman animal whose life is ended through an act that legitimately qualifies as euthanasia is normally a sentient domestic animal. On the other, the painless and merciful nature of the termination of a nonhuman animal’s life is a necessary but not sufficient condition for it to count as a genuine instance of nonhuman animal euthanasia.

343 Preparing a quality animal care and use protocol

Abstract Objectives in preparing a quality animal care and use protocol (AUP) revolve around the creation of a document that meets federal and institutional regulatory guidelines and that provides adequate detail for members of the institutional animal care and use committee to determine whether animal care and use procedures are justified and humane. Generally, either the Public Health Service Policy on Humane Care and Use of Laboratory Animals or Guide for the Care and Use of Agricultural Animals in Research and Teaching will serve as a guide for preparing AUPs. In addition, AUPs are publicly accessible; therefore, a section of the document must be written such that the lay public can easily read and understand what will happen to the animals and why. Well-written, thorough protocols must include: 1) USDA/Animal Welfare Act pain/distress category; 2) signed certification that the principal investigator, co-principal investigators and all personnel whose names appear on the AUP understand and will adhere to the requirements associated with the animal procedures to be employed and accept that they are under federal and institutional regulatory authority; 3) the work with animals is restricted specifically to what is approved in the official AUP. Specific work-related sections of the AUP should include 1) names and contact information of the principal and co-principal investigators and animal care contact personnel; 2) a lay summary that describes the purpose of the work and what specifically will happen to the animals; 3) detailed description of animal procedures and experimental design, including species, numbers, age and sex of animals; 4) animal procurement and maintenance information; 5) animal transfer and euthanasia procedures; 6) pharmaceutical/chemical agents to be administered; 7) qualifications and experience of all personnel; 8) emergency contacts and procedures; 9) complete description of surgical procedures; 10) arrangements for veterinary care.

Altered prolactin and androgen receptors expression in companion rat benign mammary tumours.

Benign mammary tumours are among the most common tumours of companion rats (Rattus norvegicus domestica), as well as a major animal welfare concern and euthanasia. The first objective of this study was to evaluate the expression of oestrogen, progesterone, androgen, and prolactin receptors in neoplastic and normal mammary gland tissues and compare the expression of these receptors between groups. The second objective was to determine if the expression of these receptors in neoplastic mammary gland tissue correlates with overall survival and occurrence of an additional mass after initial mammary mass excision. The third objective was to determine if the expression of oestrogen, progesterone, androgen and prolactin receptors was associated with mammary tumor clinical parameters or with the age of the animals. Thirty-two benign mammary tumours were collected from companion rats and submitted for immunohistochemistry staining of prolactin receptor, oestrogen receptor alpha (ERa), progesterone and androgen receptors (AR). Allred score were obtained for mammary tumours (n=32) and surrounding normal mammary tissue (n=20) when present. Prolactin receptor expression increased significantly with mammary gland tumorigenesis (P < .0001), while AR expression decreased with tumorigenesis (P < .0001). Lower expression of ERa in tumor stroma was associated with shorter survival (P=.02). Hormonal receptor expression was not significantly associated with age, mass diameter, location nor likelihood of additional mass development. Further studies should investigate the effects of prolactin antagonists in a prospective study involving companion rats with benign mammary tumours.

Behavioral and corticosterone responses to carbon dioxide exposure in reptiles

The use of carbon dioxide (CO2) exposure as a means of animal euthanasia has received considerable attention in mammals and birds but remains virtually untested in reptiles. We measured the behavioral responses of four squamate reptile species (Homalopsis buccata, Malayopython reticulatus, Python bivitattus, and Varanus salvator) to exposure to 99.5% CO2 for durations of 15, 30, or 90 minutes. We also examined alterations in plasma corticosterone levels of M. reticulatus and V. salvator before and after 15 minutes of CO2 exposure relative to control individuals. The four reptile taxa showed consistent behavioral responses to CO2 exposure characterized by gaping and minor movements. The time taken to lose responsiveness to stimuli and cessation of movements varied between 240–4260 seconds (4–71 minutes), with considerable intra- and inter-specific variation. Duration of CO2 exposure influenced the likelihood of recovery, which also varied among species (e.g., from 0–100% recovery after 30-min exposure). Plasma corticosterone concentrations increased after CO2 exposure in both V. salvator (18%) and M. reticulatus (14%), but only significantly in the former species. Based on our results, CO2 appears to be a mild stressor for reptiles, but the relatively minor responses to CO2 suggest it may not cause considerable distress or pain. However, our results are preliminary, and further testing is required to understand optimal CO2 delivery mechanisms and interspecific responses to CO2 exposure before endorsing this method for reptile euthanasia.

Adult's Pet Euthanasia Experience and Complicated Grief: An Exploratory Study on the Risk Factors

Adult's Pet Euthanasia Experience and Complicated Grief: An Exploratory Study on the Risk Factors

Establishing Animal Welfare Rules of Conduct for the Portuguese Veterinary Profession-Results from a Policy Delphi with Vignettes.

Simple SummaryPromoting animal welfare is one of the basic tenets of the veterinary profession. However, it is known that the Portuguese veterinary code of conduct, established more than 25 years ago, overlooks animal welfare and fails to address issues such as the euthanasia and humane killing of animals. Using a Policy Delphi debate technique with vignettes, this study assesses the level of agreement of forty-one (out of seventy) purposively selected Portuguese veterinarians on ten animal welfare requirements set forth in a code of professional conduct, meant to be approved in a near future. Most participants agreed that the suggested animal welfare rules of conduct reflected their own views on the subject (88%), in addition to representing a significant improvement in terms of regulatory standards (93%). This study will support regulation and policy-making by the Portuguese Veterinary Order and by veterinary representative bodies elsewhere.Promoting animal welfare is one of the basic tenets of the veterinary profession and, in doing so, veterinarians are expected to abide to the highest legal and professional standards. However, the Portuguese veterinary code of conduct, established in 1994, largely overlooks animal welfare and fails to address issues such as the euthanasia or humane killing of animals. As part of a wider research aiming to revise the Portuguese veterinary code of conduct, a Policy Delphi study was conducted in late 2018, using a pre-validated three-round structure and vignette methodology, to explore the range of opinions and the level of agreement on end-of-life dilemmas and animal welfare rules of conduct of a purposeful sample of forty-one (out of seventy) Portuguese veterinarians. When faced with ethical vignettes involving end-of-life dilemmas, veterinarians were shown to privilege personal moral agency over legal obligations in order to defend the interests of stakeholders, namely of the animals. Most participants agreed that the suggested animal welfare rules of conduct reflected their own views on the subject (88%), in addition to representing a significant improvement in terms of regulatory standards (93%). We expect that this study will support regulation and policy-making by the Portuguese Veterinary Order and by veterinary representative bodies elsewhere.

Short- and Long-Term Repeated Forced Swim Stress Induce Depressive-Like Phenotype in Mice: Effectiveness of 3-[(4-Chlorophenyl)Selanyl]-1-Methyl-1H-Indole.

Exposure to stress highly correlates with the emergence of mood-related illnesses. Therefore, the present study was designed to characterize the acute and chronic effects of 3-((4-chlorophenyl)selanyl)-1-methyl-1H-indole (CMI) on depressive-like behavior induced by repeated forced swim stress (FSS) in male adult Swiss mice. In the repeated FSS, mice were placed in water to swim for a single trial during a 15-min period. Twenty-four hours after the first FSS, the animals were placed in water to swim through a series of four trials, and each of them swam for 6 min long; between each trial, mice were towel dried and returned to their home cage for 6 min. In addition, the oxidative stress in the prefrontal cortex and hippocampus and corticosterone levels of plasma of mice were investigated. The animals exposed to FSS were treated with CM in two different protocols. In protocol 1, CMI [1 and 10 mg/kg, intragastric (i.g.) route] or fluoxetine, a positive control (10 mg/kg, i.g. route), were administered 30 min before of sections of repeated FSS in both days of stress. After the last section of repeated FSS, the mice performed first the spontaneous locomotor activity and after the tail suspension test. In protocol 2, CMI or fluoxetine (1 mg/kg, i.g. route) was administered for 20 days after the exposition of repeated FSS. The spontaneous locomotor activity, tail suspension, and forced swimming tests were performed in this order after 24 h of last administration of CMI or fluoxetine. The euthanasia of animals was performed after the behavioral tests. CMI and fluoxetine abolished the depressive-like behavior induced by repeated FSS in mice in the two different treatments. CMI modulated the oxidative stress in the prefrontal cortices and hippocampi of mice subjected to repeated FSS. Mice subjected to repeated FSS had an increase in the corticosterone levels and CMI regulated the levels of this glucocorticoid. These findings demonstrate that CMI was effective to abolish the depressive-like behavior induced by repeated FSS, which was accompanied by changes in the corticosterone levels and oxidative stress of prefrontal cortices and hippocampi of mice.

Efeito da atividade física em ambiente enriquecido sobre o músculo esquelético de camundongos com caquexia associado ao melanoma cutâneo

OBJETIVO: O objetivo deste estudo foi verificar os efeitos da atividade física em ambiente enriquecido sobre histomorfometria do músculo quadríceps femoral de camundongos C57BL/6 com caquexia associada ao modelo tumoral singênico de melanoma cutâneo. MÉTODOS: Foram utilizados camundongos fêmeas da linhagem C57BL/6 que foram distribuídos aleatoriamente em dois grupos: controle CRC (n=11), que não realizou nenhum tipo de intervenção, o segundo grupo foi o experimental (CRC-ATF) (n=15), que realizou atividade física organizada em ambiente enriquecido de 60 cm de comprimento, 30 cm de largura e 45 cm de altura. Ocorreu indução tumoral de células B16-F10 da linhagem de melanoma cutâneo em todos os animais desse estudo. Após dez dias da indução tumoral, todos os animais já apresentavam quadro de caquexia, assim o grupo experimental iniciou a atividade física em ambiente enriquecido com duração de 30 minutos com intervalos de 48 horas. Após 26 dias do início da intervenção, os animais sobreviventes foram eutanasiados e foi realizada a coleta do músculo quadríceps femoral para análise histomorfométrica. RESULTADOS: Analisando a intervenção realizada, percebemos que o grupo CRC-ATF apresentou a área da fibra muscular maior que o grupo CRC, mas esse resultado não mostrou diferença significativa (p?0,05) entre os grupos. CONCLUSÃO: Os resultados mostram que é possível preservar a estrutura muscular, pois os melhores resultados foram encontrados com os animais que participaram da atividade física em ambiente enriquecido.

The Protective Effect of Xanthohumol on the Content of Selected Elements in the Bone Tissue for Exposed Japanese Quails to TCDD.

Dioxins (including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are highly toxic and persistent chemicals widely distributed in the environment in trace amounts, and are side products of industrial and chemical processes. Exposure to dioxins leads to multiorgan morphological and functional abnormalities, including within the bone tissue, disrupting its microarchitecture and mechanical properties. Xanthohumol (XN) is a chemical compound classified as a prenylated flavonoid, distinguished by multidirectional biological action. The aim of the study is to assess whether xanthohumol, as a substance with strong antioxidant and anti-inflammatory properties, has the ability to eliminate the negative effects of TCDD on bone tissue. The experiment was conducted on adult Japanese quails. Two different doses of TCDD and xanthohumol were administered to birds. After euthanasia of animals, the research material in the form of cranial vault and hind limb bone was collected, and their mineral compositions of calcium, phosphorus, magnesium, zinc, and iron concentrations were determined using atomic emission spectrometry in an acetylene-air flame method. Our results indicate that the administration of TCDD at a low dose causes more dynamic changes in the concentration of elements in bone, in comparison to a higher dose of dioxin. Results show also that higher doses of the XN cause the linear increase in the concentration of phosphorus and iron in the bone of the hind limb, and calcium in the bones of the cranial vault. In conclusion, our experiment shows that the use of TCDD and XN in Japanese quails together in various doses influences the content of phosphorus, magnesium, zinc, and iron in the research material.

A National Survey of Companion Animal Owners' Self-Reported Methods of Coping Following Euthanasia.

(1) Background: The human–animal bond is often regarded as a special relationship in which owners benefit from unconditional love and perceived understanding from their companion animal. Thus, end-of-life decisions such as euthanasia may inflict significant emotional impact upon the companion animal owner and result in a complicated grief response. The purpose of this study was to examine the methods American companion animal owners utilize to cope with loss following companion animal euthanasia. (2) Methods: A total of 340 companion animal owners with experience euthanizing a companion animal completed an online survey asking how they found comfort after the loss of their companion animal. (3) Results: A total of 74.7% noted that they mourned privately, 58.2% sought social support, 32.1% adopted a new companion animal, 12.4% relied on faith or prayer and 0.9% participated in a support group. (4) Conclusions: Grief associated with companion animal loss is important. As a majority of clients that euthanized their companion animal mourn privately, the veterinary community must work towards identifying and providing appropriate, accessible social resources for bereaved companion animal owners to utilize, if desired.

Exploring pet owners' experiences and self-reported satisfaction and grief following companion animal euthanasia.

While euthanasia is a common feature of veterinary practice, research has yet to adequately explore the experiences, perception and wishes of pet owners, including their satisfaction and grief following companion animal euthanasia. An online questionnaire was conducted with pet owners who had experienced euthanasia within the last 10 years to explore the relationship between pet owners' experiences and their resulting satisfaction and grief following companion animal euthanasia. Data were analysed using descriptive statistics and multivariable linear regression. Overall, participants (N=2354) reported high levels of satisfaction with their euthanasia experience. Their experience with the administration practices (i.e., payment and paperwork), emotional support, follow-up care and care for their pet's remains was found to be associated with overall satisfaction. Participants' grief was associated with the number of euthanasia previously experienced, the type of human-animal bond, if the euthanasia was emergent and the emotional support they received. Findings contribute to existing research and shed light on some of the most important practices associated with companion animal euthanasia. Several practical recommendations are made, including developing standard operating procedures for companion animal euthanasia; exploring owners' previous experiences, expectations and emotions; the importance of reassurance; and access to grief resources and services.

Anti-Cancer Activity of PAK4/NAMPT Inhibitor and Programmed Cell Death Protein-1 Antibody in Kidney Cancer.

Kidney cancer (or renal cell carcinoma, RCC) is the sixth most common malignancy in the United States and is increasing in incidence. Despite new therapies, including targeted therapies and immunotherapies, most RCCs are resistant to treatment. Thus, several laboratories have been evaluating new approaches to therapy, both with single agents as well as combinations. Although we have previously shown efficacy of the dual PAK4/nicotinamide phosphoribosyltransferase (NAMPT) inhibitor KPT-9274, and the immune checkpoint inhibitors (CPI) have shown utility in the clinic, there has been no evaluation of this combination either clinically or in an immunocompetent animal model of kidney cancer. In this study, we use the renal cell adenocarcinoma (RENCA) model of spontaneous murine kidney cancer. Male BALB/cJ mice were injected subcutaneously with RENCA cells and, after tumors were palpable, they were treated with KPT-9274 and/or anti-programmed cell death 1 (PDCD1; PD1) antibody for 21 days. Tumors were measured and then removed at animal euthanasia for subsequent studies. We demonstrate a significant decrease in allograft growth with the combination treatment of KPT-9274 and anti-PD1 antibody without significant weight loss by the animals. This is associated with decreased (MOUSE) Naprt expression, indicating dependence of these tumors on NAMPT in parallel to what we have observed in human RCC. Histology of the tumors showed substantial necrosis regardless of treatment condition, and flow cytometry of antibody-stained tumor cells revealed that the enhanced therapeutic effect of KPT-9274 and anti-PD1 antibody was not driven by infiltration of T cells into tumors. This study highlights the potential of the RENCA model for evaluating immunologic responses to KPT-9274 and checkpoint inhibitor (CPI) and suggests that therapy with this combination could improve efficacy in RCC beyond what is achievable with CPI alone.

Laboratory Mice Euthanasia: Speed Death and Animal Welfare

The euthanasia is one of the most studied, debated and controversial topics in the laboratory animal science area. All over the world, the ideal way to perform euthanasia in laboratory animals is sought, seeking, in summary, three main points, animal welfare during the procedure; minimal influence on the animals biological system, not to interfere with test results and accessibility and low cost. In Brazil, after the validity of Law No 11.794-2008, that regulated the use of animals for didactic and scientific purposes, structuring the Animal Experimentation Control Council (CONCEA). In its resolutions, CONCEA standardized the application of accepted euthanasia methods, mainly overdose of injectable anesthetics and restricted use of carbon dioxide (CO2).

Exploring veterinarians’ use of practices aimed at understanding and providing emotional support to clients during companion animal euthanasia in Ontario, Canada

BackgroundDuring companion animal euthanasia, support of clients is equally important as the medical care of the companion animal and requires a clear and developed understanding of clients' feelings, needs and...

Neuroprotective Effects of Co-administration of Coenzyme Q10 and Vitamin-E in Chronic Cerebral Hypoperfusion-Induced Neurodegeneration in Rats

Alzheimer’s disease (AD) is the most common type of neurodegenerative diseases. Currently, there is no prevention or cure for AD. The potential use of natural antioxidants for prevention and treatment of AD has attracted considerable attention. Here, we used combination of the antioxidants coenzyme Q10 (CoQ10) and vitamin-E (Vit E) for the protection against AD. The current study assessed the neuroprotective effects of combination of CoQ10 with Vit E in Chronic Cerebral Hypoperfusion-induced neurodegeneration (CCH-ND) rat model. After acclimatization, 27 Sprague Dawley rats weighing 220–250 g were divided into six groups; sham control, 2-vessel occlusion (2VO), 2VO+E (treated daily with Vit E, 100 mg/kg, orally following 2VO), CoQ10 (treated daily with CoQ10, 200 mg/kg, orally following 2VO), CoQ10+E (treated with combination of CoQ10 and Vit E, orally following 2VO) and last group was treated with coconut-oil as a vehicle control. On the 8th week, all rats were tested by Morris water maze cognitive test and then euthanized and the hippocampi were isolated. Viable neuronal cell count in the hippocampal region was estimated. The Isoprostane F2 (F2-IsoPs) levels were assessed in the brain homogenates to quantify the oxidative stress status. There was significant difference in neuronal cell death, memory and learning, and F2-Iso level in untreated 2VO group compared to the treated and sham groups. However, there was no statistically significant difference in neuroprotective effects of combination of Vit E with CoQ10 and each one alone. To conclude, combination of the antioxidants (Vit E and CoQ10) improves memory, neuronal cell viability and decreases antioxidant level, same as each antioxidant alone.

Guidelines for best practice in placebo-controlled experimental studies on probiotics in rodent animal models.

In the absence of established best practice standards in the probiotic field for reducing the risk of bacterial transfer between experimental groups, we developed protocols and methods to ensure the highest quality and interpretability of results from animal studies, even when performed in non-conventional animal care facilities. We describe easily implementable methods for reducing cross-contamination during animal housing, behavioural testing, and euthanasia, along with highlighting protocols for contamination detection in experimental subjects and laboratory areas using qPCR. In light of the high cross-contamination risks between animals during experiments involving probiotics, constant vigilance in animal care and research protocols is critical to ensure valid and reliable research findings.

Identification of Black Bullhead (Ameiurus melas) and Brown Bullhead (A. nebulosus) from the Western Great Lakes: Recommendations for Small Individuals

Distinguishing Black Bullhead (Ameiurus melas) from Brown Bullhead (A. nebulosus) can be difficult, in part, due to conflicting evidence regarding reliable meristic and morphological characters with which to base identifications. The possibility of hybrids with intermediate traits further complicates diagnosis. Identification guides have typically focused on adults, leaving guidance for juveniles largely undescribed. We compared morphological identifications of Black Bullhead and Brown Bullhead with a cytochrome c oxidase I (COI) barcode and microsatellite assignments. Specifically, we examined: (1) gill-raker count, (2) anal-fin ray count, (3) prominence of pectoral-spine serrae, (4) DNA barcoding using the COI gene, and (5) cluster analysis of nine microsatellite loci. With the exception of anal-fin ray counts, we found a high degree of agreement among all methods. Microsatellite analysis identified two clearly distinct genetic groups, and these groups corresponded to individuals identified as Black Bullhead and Brown Bullhead based on morphology. Although Black Bullhead and Brown Bullhead did not share COI haplotypes (except for one individual), we found greater intraspecific genetic distances among individuals identified as Brown Bullhead than between Brown Bullhead and Black Bullhead, demonstrating that caution is warranted against using COI haplotypes as a barcode to solely distinguish the two species. Pectoral-spine serrae prominence was a useful character to distinguish the two species and worked especially well on individuals <125 mm total length. Gill-raker counts of euthanized fish in the laboratory were appropriate for the identification of all sizes of Black Bullhead and Brown Bullhead (using the lowest number of gill rakers on the first gill arch if asymmetry was observed). Finally, microsatellites did not show evidence of recent hybridization at our sampling sites. We recommend using pectoral-spine serrae prominence to distinguish between Black Bullhead and Brown Bullhead in the field and using gill-raker counts for laboratory based identification.