scholarly journals Refinement of intraperitoneal injection of sodium pentobarbital for euthanasia in laboratory rats (Rattus norvegicus)

2016 ◽  
Author(s):  
K Zatroch ◽  
CG Knight ◽  
JN Reimer ◽  
DSJ Pang
Keyword(s):  
Coefficient Of Variation ◽  
Low Dose ◽  
High Volume ◽  
High Dose ◽  
Sodium Pentobarbital ◽  
Sprague Dawley ◽  
Loss Of Righting Reflex ◽  
Canadian Council ◽  
Righting Reflex ◽  
Low Volume

AbstractBackgroundThe Canadian Council on Animal Care and American Veterinary Medical Association classify intraperitoneal (IP) pentobarbital as an acceptable euthanasia method in rats. However, federal guidelines do not exist for a recommended dose or volume and IP euthanasia has been described as unreliable, with misinjections leading to variable success in ensuring a timely death. The aims of this study were to assess and improve efficacy and consistency of IP euthanasia.MethodsIn a randomized, blinded study, 51 adult female Sprague-Dawley rats (170-495 g) received one of four treatments: low-dose low-volume (LL) IP pentobarbital (n = 13, 200 mg/kg pentobarbital), low-dose high-volume (LH) IP pentobarbital (n = 14, 200 mg/kg diluted 1:3 with phosphate buffered saline), high-dose high-volume (HH, n = 14, 800 mg/kg pentobarbital), or saline. Times to loss of righting reflex (LORR) and cessation of heartbeat (CHB) were recorded. To identify misinjections, necropsy examinations were performed on all rats. Video recordings of LL and HH groups were analyzed for pain-associated behaviors. Between-group comparisons were performed with 1-way ANOVA and Games-Howell post hoc tests. Variability for CHB was assessed by coefficient of variation (CV) calculation.ResultsThe fastest euthanasia method (CHB) was HH (283.7 ± 38 s), compared with LL (485.8 ± 140.7 s, p = 0.002) and LH (347.7 ± 72.0 s, p = 0.039). Values for CV were: HH, 13.4%; LH, 20.7%; LL, 29.0%. LORR time was longest in LL (139.5 ± 29.6 s), compared with HH (111.6 ± 19.7 s, p = 0.046) and LH (104.2 ± 19.3 s, p = 0.01). Misinjections occurred in 15.7% (8/51) of euthanasia attempts. Pain-associated behavior incidence ranged from 36% (LL) to 46% (HH).ConclusionThese data illustrate refinement of this euthanasia technique. Both dose and volume contribute to speed of death with IP pentobarbital and an increase in volume alone does not significantly reduce variability. The proportion of misinjections was similar to that of previous studies.AbbreviationsLLlow-dose low-volumeLHlow-dose high-volumeHHhigh-dose high-volumeLORRloss of righting reflexCHBcessation of heartbeatGITgastrointestinal tractCVcoefficient of variation

scholarly journals Intraperitoneal injection of sodium pentobarbital is associated with pain in rats

2019 ◽  
Author(s):  
JN Reimer ◽  
C Schuster ◽  
CG Knight ◽  
DSJ Pang ◽  
VSY Leung
Keyword(s):  
Relative Frequency ◽  
Behavioral Assessment ◽  
Indirect Evidence ◽  
Vehicle Control ◽  
Control Group ◽  
Sodium Pentobarbital ◽  
Sprague Dawley ◽  
Control Groups ◽  
Loss Of Righting Reflex ◽  
Righting Reflex

AbstractAn effective and pain-free killing method is required to achieve the goal of euthanasia, a “good death”. Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely accepted technique, but questions remain regarding pain associated with administration. As PB rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence of pain. The objective of this study was to assess pain associated with IP PB using an appropriate vehicle control.Adult male and female Sprague Dawley (SD) and female Wistar rats (N = 112) were block randomised by sex and strain to receive one of four treatments: 1) 800 mg/kg PB (pH 11); 2) 800 mg/kg PB with 4 mg/kg lidocaine (PB+lido); 3) saline or 4) vehicle controls (pH 11 or 12.5). Behavior (Rat Grimace Scale [RGS], writhing, back arching) was evaluated at baseline, before loss of righting reflex (PB and PB+lido groups), 80s, 151s and 10 min post-injection (PI; saline and vehicle control groups).In the vehicle control groups, the RGS scores were increased at 151s PI (SD: p = 0.0008, 95%CI −0.731 to −0.202) from baseline, as was relative frequency of writhing (SD: p < 0.00001; Wistar; p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0070, 95%CI −0.768 to −0.118; Wistar: p = 0.0236, 95%CI −0.907 to −0.0742) but the relative frequency of writhing did not (p > 0.05). The RGS scores and the relative frequency of writhing remained low in the PB, PB+lido and saline groups (p > 0.05). Back arching increased from baseline in the PB+lido group before loss of righting reflex and in the vehicle control group (SD rats) at 151s PI (p < 0.05).These results show that IP PB results in signs associated with pain. The sedative effects of PB limit behavioral assessment.

scholarly journals Relationships among Dissemination of Primary Parainfluenza Virus Infection in the Respiratory Tract, Mucosal and Peripheral Immune Responses, and Protection from Reinfection: a Noninvasive Bioluminescence-Imaging Study

2015 ◽  
Vol 89 (7) ◽  
pp. 3568-3583 ◽  
Author(s):  
Crystal W. Burke ◽  
Mei Li ◽  
Julia L. Hurwitz ◽  
Peter Vogel ◽  
Charles J. Russell
Keyword(s):  
Immune Responses ◽  
Primary Infection ◽  
Low Dose ◽  
Bioluminescence Imaging ◽  
High Volume ◽  
Antibody Responses ◽  
High Dose ◽  
Lethal Challenge ◽  
Contact Transmission ◽  
Low Volume

ABSTRACTRespiratory paramyxoviruses such as respiratory syncytial virus (RSV) and human parainfluenza virus type 1 (HPIV1) to HPIV4 infect virtually all children by the age of 2 to 5 years, leading to partial but incomplete protection from reinfection. Here, we used luciferase-expressing reporter Sendai viruses (the murine counterpart of HPIV1) to noninvasively measure primary infection, immune responses, and protection from reinfection by either a lethal challenge or natural transmission in living mice. Both nonattenuated and attenuated reporter Sendai viruses were used, and three inoculation strategies were employed: intramuscular (i.m.), intranasal (i.n.) at a low dose and low volume, and i.n. at a high dose and high volume. High-dose, high-volume i.n. inoculation resulted in the highest levels of antibody responses and protection from reinfection. Low-dose, low-volume i.n. inoculation afforded complete protection from contact transmission and protection from morbidity, mortality, and viral growth during lethal challenge. i.m. inoculation was inferior to i.n. inoculation at inducing antibody responses and protection from challenge. For individual mice and across groups, the levels of serum binding and neutralizing antibody responses correlated with primary infection and protection from reinfection in the lungs. Contact transmission, the predominant mode of parainfluenza virus transmission, was modeled accurately by direct i.n. inoculation of Sendai virus at a low dose and low volume and was completely preventable by i.n. vaccination of an attenuated virus at a low dose and low volume. The data highlight differences in infection and protection from challenge in the upper versus lower respiratory tract and bear upon live attenuated vaccine development.IMPORTANCEThere are currently no licensed vaccines against HPIVs and human RSV (HRSV), important respiratory pathogens of infants and children. Natural infection leads to partial but incomplete protective immunity, resulting in subsequent reinfections even in the absence of antigenic drift. Here, we used noninvasive bioluminescence imaging in a mouse model to dissect relationships among (i) the mode of inoculation, (ii) the dynamics of primary infection, (iii) consequent immune responses, and (iv) protection from high-dose, high-volume lethal challenge and contact transmission, which we find here to be similar to that of a mild low-dose, low-volume upper respiratory tract (URT)-biased infection. Our studies demonstrate the superiority of i.n. versus i.m. vaccination in protection against both lethal challenge and contact transmission. In addition to providing correlates of protection that will assist respiratory virus vaccine development, these studies extend the development of an increasingly used technique for the study of viral infection and immunity, noninvasive bioluminescence imaging.

Ferulic Acid Dose Effect on Pharmacokinetics of Glimepiride and its Metabolite Hydroxy Glimepiride in Rats

2021 ◽  
Vol 17 ◽  
Author(s):  
Yuxian Lin ◽  
Faxin Sun ◽  
Jinlai Liu ◽  
Qinghua Weng ◽  
Lijun Jin ◽  
...  
Keyword(s):  
Ferulic Acid ◽  
Low Dose ◽  
Dose Effect ◽  
High Dose ◽  
Intragastric Administration ◽  
Healthy Male ◽  
Sprague Dawley ◽  
Sd Rats ◽  
High Dose Treatment ◽  
Significant Difference

Background: To mitigate diabetes and its complications in cardiovascular diseases, the antidiabetic agent glimepiride is usually administered with ferulic acid concomitantly in clinics. However, both drugs are prone to be metabolized partly by CYP2C9, thus they have the potential drug-drug interaction affecting the safety and efficacy. Objective: This project aimed to evaluate the pharmacokinetic (PK) effects of ferulic acid (FA) on glimepiride (GLM) and its metabolite hydroxy glimepiride (OH-GLM) in plasma by using the HPLC-MS/MS method. Methods: Healthy male Sprague Dawley (SD) rats were randomly divided into three groups. They received intragastric administration of 0.5% sodium carboxymethyl cellulose (CMC), low-dose FA (20 mg•kg-1), and high-dose FA (40 mg•kg-1) for 8 days, respectively. Rats were given 0.5% sodium CMC or FA on the last day and then uniformly given 1.0 mg•kg-1 glimepiride by gavage. Blood samples were obtained from retro-orbital plexus at the time points of 0.167, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after administration. Plasma samples were analyzed for GLM and its metabolite OH-GLM on an HPLC-MS/MS system. Results: No statistically significant difference was found in the effect of low-dose FA on the pharmacokinetics of GLM. High-dose FA significantly decreased Cmax of GLM by 30.05% and CLz/F of OH-GLM by 47.45%. It also increased Tmax and t1/2z of GLM by 95.87% and 140.00%. Conclusion: Low-dose FA did not alter GLM metabolism, while high-dose treatment of FA caused pharmacokinetics interaction with GLM in rats.

scholarly journals Attenuation of Thrombosis by Crude Rice (Oryza sativa) Bran Policosanol Extract:Ex VivoPlatelet Aggregation and Serum Levels of Arachidonic Acid Metabolites

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Wai-Teng Wong ◽  
Maznah Ismail ◽  
Eusni Rahayu Mohd Tohit ◽  
Rasedee Abdullah ◽  
Yi-Da Zhang
Keyword(s):  
Platelet Aggregation ◽  
Rice Bran ◽  
Low Dose ◽  
Ex Vivo ◽  
Vascular Occlusion ◽  
Full Blood Count ◽  
Tween 20 ◽  
High Dose ◽  
Sprague Dawley ◽  
Coagulation Time

Background. Vascular occlusion or thrombosis was often attributed to uncontrolled platelet activation. Influence of sugarcane policosanol extract on platelet was reported but little was known of rice bran policosanol, particularly its mechanisms of actions on platelet activities.Objective. Antiplatelet mechanisms of rice bran policosanol extract (RBE) were studied using hyperlipidemic Sprague Dawley rats.Ex vivoplatelet aggregation, platelet count (PC), bleeding time (BT), and coagulation time were assayed. Serum eicosanoids and other aggregation-related metabolites levels were quantified.Design. Rats were divided into 6 groups for comparisons (vehicle control Tween 20/H2O, high dose policosanol 500 mg/kg, middle dose policosanol 250 mg/kg, low dose policosanol 100 mg/kg, and positive control aspirin 30 mg/kg).Results. Low dose 100 mg/kg of RBE inhibited aggregation by42.32±4.31% and this was comparable with the effect of 30 mg/kg aspirin,43.91±5.27%. Results showed that there were no significant differences in PC, BT, and coagulation time among various groups after RBE treatment. Serum thromboxane A2was attenuated while prostacyclin level increased upon RBE treatment.Conclusions. RBE reducedex vivoADP-induced platelet aggregation without giving adverse effects. No changes in full blood count suggested that rice bran policosanol did not disturb biological blood cell production and destruction yet it reduced aggregation through different mechanisms.

scholarly journals Original Article. Inflammatory effect of 2-aminoanthracene (2AA) on adipose tissue gene expression in pregnant Sprague Dawley rats

2016 ◽  
Vol 9 (1) ◽  
pp. 17-24
Author(s):  
Shamaya L. Whitby ◽  
Daniel A. Hunter ◽  
Wilson Yau ◽  
Elizabeth W. Howerth ◽  
Worlanyo E. Gato
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Dose Group ◽  
Low Dose ◽  
Mononuclear Cells ◽  
Postnatal Period ◽  
High Dose ◽  
Sprague Dawley ◽  
Polycyclic Aromatic ◽  
Altered Gene

Abstract Adipocyte dysfunction may be a critical link between obesity and insulin resistance as a result of abnormal fat storage and mobilization. Adipocytes uniquely secrete adipokines and cytokines, such as leptin and TNFα, wich promote insulin sensitivity. Previously we reported insulin-signaling related altered gene expression in animals exposed to 2-Aminoanthracene (2AA). 2AA is an aminosubstituted polycyclic aromatic hydrocarbon used in manufacturing dyes, chemicals, inks, resins, and polyurethanes. The objective of this study was to examine the inflammation related effects of 2AA exposure from gestation to postnatal period on dams that ingested 2AA. To examine 2AA effects, pregnant dams were assigned into dose regimens of 2AA. Dams were fed 2AA contaminated diet during the period of gestation and postpartum. The expression of key gene transcripts reported to be important in mediating inflammatory processes was examined via quantitative RT-PCR. Histologic examination of adipose tissue (AT) was also carried out to understand the anatomy of AT due to 2AA exposure during gestation and two weeks postpartum. Examination of the adipose tissue for microscopic changes revealed no alterations between control and low-dose animals. However, AT of the high-dose animals was infiltrated by increased numbers of CD68+mononuclear cells (macrophages) and small numbers of eosinophils and mast cells, consistent with inflammation. In addition, analysis of the mRNA expression of cytokines and adipokines demonstrated the importance of inflammation in AT dysfunction. For instance, TNFα, LEPTIN and IL-6 transcripts were relatively more expressed in the low dose animals than in the high dose and control rats. At the protein level, however, high amounts of cytokines were noted. The effects of 2AA on pregnant dams appear to be more pronounced in the high dose group than in the low dose group, possibly indicating increased susceptibility of rat offspring within this group to elicit a diabetic-type response.

scholarly journals Blood Biochemical and Hematological Study after Subacute Intravenous Injection of Gold and Silver Nanoparticles and Coadministered Gold and Silver Nanoparticles of Similar Sizes

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Ji Hyun Lee ◽  
Mary Gulumian ◽  
Elaine M. Faustman ◽  
Tomomi Workman ◽  
KiSoo Jeon ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Low Dose ◽  
Systemic Toxicity ◽  
Blood Biochemistry ◽  
Control Group ◽  
High Dose ◽  
Sprague Dawley ◽  
Gold And Silver Nanoparticles ◽  
Blood Biochemical ◽  
Active Partial Thromboplastin Time

Background. To investigate the effect of subacute intravenous administration AgNP (silver nanoparticles, 10 nm) and AuNP (gold nanoparticles, 12.8 nm) and AgNP/AuNP mixture to blood biochemistry, hematology, and platelet coagulation, subacute toxicity study was conducted. Methods. AuNP and AgNP in which their size distribution was not statistically different, mixed or separate, were injected into the caudal vein of male Sprague-Dawley rats for 4 weeks. The rats were allowed to recover for a further 4 weeks in order to examine systemic toxicity expressed in the blood biochemistry and hematology. The dose groups (5 males per group for the administration and 3 males for the recovery) consisted of 7 divisions, i.e., control, AgNP (with a low dose of 10 μg/kg/day and a high dose of 100 μg/kg/day), AuNP (with a low dose of 10 μg/kg/day and a high dose of 100 μg/kg/day), and mixed AgNP/AuNP (with a low dose of 10/10 μg/kg/day and a high dose of 100/100 μg/kg/day). Results. There were no significant dose-related changes in the hematology and blood biochemical values for the rats. Coagulation time in terms of the active partial thromboplastin time (APTT) and prothrombin time (PT) did not show any significant changes, when compared to the control group. Conclusion. The subacute injection of AuNP and AgNP or their mixture did not induce any noticeable systemic toxicity.

Sympathetic and parasympathetic component of bradycardia triggered by stimulation of NTS P2X receptors

2006 ◽  
Vol 290 (2) ◽  
pp. H807-H812 ◽  
Author(s):  
Amy M. Kitchen ◽  
Donal S. O'Leary ◽  
Tadeusz J. Scislo
Keyword(s):  
Low Dose ◽  
Nucleus Tractus Solitarius ◽  
Receptor Activation ◽  
P2x Receptors ◽  
P2x Receptor ◽  
High Dose ◽  
Adrenergic Blockade ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Stimulation Of

We have previously shown that activation of P2X purinoceptors in the subpostremal nucleus tractus solitarius (NTS) produces a rapid bradycardia and hypotension. This bradycardia could occur via sympathetic withdrawal, parasympathetic activation, or a combination of both mechanisms. Thus we investigated the relative roles of parasympathetic activation and sympathetic withdrawal in mediating this bradycardia in chloralose-urethane anesthetized male Sprague-Dawley rats. Microinjections of the selective P2X purinoceptor agonist α,β-methylene ATP (25 pmol/50 nl and 100 pmol/50 nl) were made into the subpostremal NTS in control animals, after atenolol (2 mg/kg iv), a β1-selective antagonist, and after atropine methyl bromide (2 mg/kg iv), a muscarinic receptor antagonist. The bradycardia observed with activation of P2X receptors at the low dose of the agonist is mediated almost entirely by sympathetic withdrawal. After β1-adrenergic blockade, the bradycardia was reduced to just −5.1 ± 0.5 versus −28.8 ± 5.1 beats/min in intact animals. Muscarinic blockade did not produce any significant change in the bradycardic response at the low dose. At the high dose, both β1-adrenergic blockade and muscarinic blockade attenuated the bradycardia similarly, −37.4 ± 6.4 and −40.6 ± 3.7 beats/min, respectively, compared with −88.0 ± 11 beats/min in control animals. Double blockade of both β1-adrenergic and muscarinic receptors virtually abolished the response (−2.5 ± 0.8 beats/min). We conclude that the relative contributions of parasympathetic activation and sympathetic withdrawal are dependent on the extent of P2X receptor activation.

scholarly journals Inactivation of Prefrontal Cortex Delays Emergence From Sevoflurane Anesthesia

2021 ◽  
Vol 15 ◽  
Author(s):  
Emma R. Huels ◽  
Trent Groenhout ◽  
Christopher W. Fields ◽  
Tiecheng Liu ◽  
George A. Mashour ◽  
...  
Keyword(s):  
Prefrontal Cortex ◽  
Brain Regions ◽  
Association Cortex ◽  
Sprague Dawley ◽  
Subcortical Structures ◽  
Behavioral Arousal ◽  
Barrel Field ◽  
Loss Of Righting Reflex ◽  
Parietal Association Cortex ◽  
Righting Reflex

Studies aimed at investigating brain regions involved in arousal state control have been traditionally limited to subcortical structures. In the current study, we tested the hypothesis that inactivation of prefrontal cortex, but not two subregions within parietal cortex—somatosensory barrel field and medial/lateral parietal association cortex—would suppress arousal, as measured by an increase in anesthetic sensitivity. Male and female Sprague Dawley rats were surgically prepared for recording electroencephalogram and bilateral infusion into prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9). After at least 10 days of post-surgical recovery, 156 μM tetrodotoxin or saline was microinjected into one of the cortical sites. Ninety minutes after injection, rats were anesthetized with 2.5% sevoflurane and the time to loss of righting reflex, a surrogate for loss of consciousness, was measured. Sevoflurane was stopped after 45 min and the time to return of righting reflex, a surrogate for return of consciousness, was measured. Tetrodotoxin-mediated inactivation of all three cortical sites decreased (p &lt; 0.05) the time to loss of righting reflex. By contrast, only inactivation of prefrontal cortex, but not somatosensory barrel field or medial/lateral parietal association cortex, increased (p &lt; 0.001) the time to return of righting reflex. Burst suppression ratio was not altered following inactivation of any of the cortical sites, suggesting that there was no global effect due to pharmacologic lesion. These findings demonstrate that prefrontal cortex plays a causal role in emergence from anesthesia and behavioral arousal.

Neuroimmunological Effects of Exposure to Methylmercury Forms in the Sprague-Dawley Rats. Activation of the Hypothalamic-Pituitary-Adrenal Axis and Lymphocyte Responsiveness

1997 ◽  
Vol 13 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Hector G. Ortega ◽  
Manuel Lopez ◽  
Atsushi Takaki ◽  
Qin-Heng Huang ◽  
Akira Arimura ◽  
...  
Keyword(s):  
Hpa Axis ◽  
Low Dose ◽  
Fold Increase ◽  
Exposed Group ◽  
Blood Levels ◽  
High Dose ◽  
Sprague Dawley ◽  
Short Term ◽  
Hypothalamic Pituitary Adrenal ◽  
Short Term Exposure

The effects of different methylmercury (MeHg) forms on the immune system and the hypothalamic pituitary adrenal (HPA) axis were assessed. The lymphocyte response to Concanavalin A (Con A) stimulation, blood levels of interleukin-6 (IL-6), adrenocorticotrophin hormone (ACTH), and corticosterone in the presence of different MeHg compounds was measured. Rats were exposed to methylmercury sulfide [(MeHg)2S] and methylmercury chloride (MeHgCl) at concentrations of 5 and 500 μg per liter in the drinking water for 8 or 16 weeks. Short-term exposure (8 weeks) at both, low- and high-doses of (MeHg)2S significantly enhanced lymphocyte responsiveness. MeHgCl only induced increased lymphocyte responsiveness at the low-dose exposure. Circulating levels of IL-6 after short-term exposure were increased in the MeHgCl-exposed group. The HPA axis activation was demonstrated by increased levels of ACTH and corticosterone levels. This response was predominant in low-dose exposed animals. Long-term (16 weeks) exposure resulted in a reduction in lymphocyte proliferation after both low- and high-dose MeHgCl exposures. The (MeHg)2S exposure resulted in a 3-fold increase in the proliferative response. Levels of ACTH were elevated 3-fold in the (MeHg)2S-exposed group, and no increase of corticosterone was observed in the high-dose exposed group at 8 weeks, no effect of(MeHg)2S was observed at 16 weeks. The MeHgCl exposed group showed an increase in ACTH and corticosterone levels at 8 weeks; this response was not observed at 16 weeks. These data indicate that exposure to MeHg compounds enhances T-cell proliferation in most of the cases, in a dose- and time-dependent fashion. Release of IL-6 also depends on the length of exposure. Early increases in circulating ACTH at 8 weeks also suggest activation of the HPA axis. This may contribute to the production of IL-6 and surveillance of regulatory homeostatic responses against environmental agents that mimic stress-like responses.

scholarly journals Assessment of carbon dioxide, carbon dioxide/oxygen, isoflurane and pentobarbital killing methods in rats

2016 ◽  
Author(s):  
Jessica M Chisholm ◽  
Daniel SJ Pang
Keyword(s):  
Carbon Dioxide ◽  
Heart Rate ◽  
Loss Of Consciousness ◽  
Sprague Dawley ◽  
Loss Of Righting Reflex ◽  
Sprague Dawley Rats ◽  
General Order ◽  
Righting Reflex ◽  
High Concentrations ◽  
The Relationship

AbstractBackground:Exposure to carbon dioxide (CO2) gas as a killing method is aversive and exposure to high concentrations likely to be painful. Bradycardia during exposure to CO2 is associated with nociception and pain. However, it is unclear if bradycardia occurs before loss of consciousness as this is variably defined in the literature. The objectives of this study were to explore the relationship between recumbency, loss of righting reflex (LORR) and a quiescent electromyograph as measures of loss of consciousness, and identify the onset of bradycardia in relation to these measures.Methods:Thirty-two adult, female Sprague-Dawley rats were instrumented with a telemetry device and randomly assigned to one of four killing methods (100% CO2, CO2 (70%)/O2 (30%), isoflurane (5%) and intraperitoneal pentobarbital (200 mg/kg). Time to achieve recumbency, LORR, quiescent electromyograph, isoelectric electrocorticograph, heart rate and apnea were recorded.Results:The general order of progression was recumbency, LORR, quiescent electromyograph, isoelectric electrocorticograph and apnea. Recumbency preceded LORR in the majority of animals (CO2; 7/8, CO2/O2; 8/8, isoflurane; 5/8, pentobarbital; 4/8). Bradycardia occurred before recumbency in the CO2 (p = 0.0002) and CO2/O2 (p = 0.005) groups, with a 50% reduction in heart rate compared to baseline. The slowest (time to apnea) and least consistent killing methods were CO2/O2 (1180 ± 658.1s) and pentobarbital (875 [239 to 4680]s).Conclusion:Bradycardia, and consequently nociception and pain, occurs before loss of consciousness during CO2 exposure. Pentobarbital displayed an unexpected lack of consistency, questioning its classification as an acceptable euthanasia method in rats.

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