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Evaluating Intrahepatic and Intraperitoneal Sodium Pentobarbital or Ethanol for Mouse Euthanasia.

ABSTRACT: Intraperitoneal (IP) injection of sodium pentobarbital (PB) is an accepted method of euthanasia for mice. However, this method has important drawbacks, including the potential for pain or misinjection. The objective of this prospective, randomized, blinded study was to determine whether intrahepatic (IH) injection of PB is more effective than IP delivery for mouse euthanasia. Secondary objectives were to: 1) determine whether IP ethanol (ET) is a suitable alternative to PB and 2) study the effect of isoflurane anesthesia on euthanasia with either PB or ET. Eighty adult CD1 mice were randomly assigned to 6 different treatment groups, were euthanized by using IP or IH injections of either PB or ET, and were either anesthetized or conscious before injection. Variables of interest were: 1) misinjection rates (based on necropsy evaluation), 2) time from injection to apnea and 3) time to cessation of heartbeat (CHB). The misinjection rate for IH injections was 93% (28/30). Two successful IH injections resulted in death within 4 s, but this method cannot be recommended due to the possibility for intrathoracic injection ( n = 4). In nonanesthetized mice, time to apnea and CHB was significantly shorter with IP ET (apnea: 72.5 s [median], CHB: 115 s) than with IP PB (apnea: 136 s, CHB: 176 s). Anesthesia at time of injection was associated with a shorter CHB time for IP PB. These data show the difficulty in achieving successful IH injections in mice, but confirm that IP ET is a viable and potentially superior alternative to IP PB. Lastly, anesthesia can shorten time to death after IP injection of PB.

SUBMITTER: Laferriere CA

PROVIDER: S-EPMC7210731 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Evaluating Intrahepatic and Intraperitoneal Sodium Pentobarbital or Ethanol for Mouse Euthanasia.

Laferriere Colin A CA Leung Vivian Sy VS Pang Daniel Sj DS

Journal of the American Association for Laboratory Animal Science : JAALAS 20200327 3

Intraperitoneal (IP) injection of sodium pentobarbital (PB) is an accepted method of euthanasia for mice. However, this method has important drawbacks, including the potential for pain or misinjection. The objective of this prospective, randomized, blinded study was to determine whether intrahepatic (IH) injection of PB is more effective than IP delivery for mouse euthanasia. Secondary objectives were to: 1) determine whether IP ethanol (ET) is a suitable alternative to PB and 2) study the effec ...[more]

PMID: 32220260

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Project description:The gamma-aminobutyric acid (GABA)(A) receptors mediating the discriminative stimulus effects of ethanol were studied by comparing the potency of ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)benzodiazepine-3-carboxylate (Ro15-4513) and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)-benzodiazepine-3-carboxylate (flumazenil, Ro15-1788) to antagonize ethanol, pentobarbital (PB), and midazolam substitution for ethanol. Ro15-4513 has high affinity for receptors containing alpha(4/6) and alpha(5) subunits and lower affinity for alpha(1), alpha(2), and alpha(3) subunits. Flumazenil is nonselective for GABA(A) receptors containing alpha(1), alpha(2), alpha(3), and alpha(5) subunits and has low affinity for alpha(4/6)-containing receptors. Male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) were trained to discriminate ethanol (1.0 or 2.0 g/kg i.g., 30-min pretreatment) from water. Ethanol, PB, and midazolam dose-dependently substituted for ethanol (80% ethanol-appropriate responding). Ro15-4513 (0.003-0.56 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in a vast majority of monkeys tested (15/15, 16/17, and 11/12, respectively). In contrast, flumazenil (0.30-10.0 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in 9 of 16, 12 of 16, and 7 of 9 monkeys tested, respectively. In the monkeys showing antagonism with both Ro15-4513 and flumazenil, ethanol and PB substitution were antagonized more potently by Ro15-4513 than by flumazenil, whereas midazolam substitution was antagonized with similar potency. There were no sex or training dose differences, with the exception that flumazenil failed to antagonize ethanol substitution in males trained to discriminate 2.0 g/kg ethanol. GABA(A) receptors with high affinity for Ro15-4513 (i.e., containing alpha(4/6) and alpha(5) subunits) may be particularly important mediators of the multiple discriminative stimulus effects of ethanol through GABA(A) receptor systems.

Project description:This study investigated changes in the electroencephalograph (EEG) power spectrum as well as physiological and behavioral responses to on-farm killing via mechanical cervical dislocation (MCD), manual cervical dislocation (CD) or intravenous pentobarbital sodium administration in lightly anesthetized laying hens, to evaluate the welfare impact of each method. A mixed group of 44 white Leghorn and Smoky Joe laying hens (60 weeks-old) were anesthetized with isoflurane in oxygen and maintained at 1.5-2% isoflurane/O2 until the killing method was applied. Birds were randomly assigned to one of three experimental groups on each trial day. The EEG was recorded bilaterally in a four-electrode montage. After recording a 5-min baseline, the killing method was applied and EEGs and other behavioral and physiological responses, including convulsions, gasping, cessation of body movements and feather erection were recorded for 5 min. Changes in EEG frequency bands (alpha, beta, delta, theta), median frequency (F50), 95% spectral edge frequency (F95), and total power (Ptot) were used to assess the quality of the on-farm killing event. Within 15 s after administration of pentobarbital sodium, there were significant decreases in mean frequency bands, increases in mean F50 and F95, and decreases in Ptot, suggesting brain death. In addition, birds presented a shorter latency to cessation of movement after pentobarbital sodium injection compared to MCD and CD (22 vs. 115 s and 136 s, respectively). There were significant increases in F95 and decreases in Ptot at 120 s after application of CD; and a concomitant decrease in the frequency bands at 135 s and isoelectric EEG at 171 ± 15 s. Changes consistent with brain death after MCD included isoelectric EEG at 207 ± 23 s and a significant decreases in some frequency bands at 300 s post-application. No other significant spectrum frequency changes were observed in the MCD group, suggesting brain death likely occurred near the 5-min endpoint. There was no clear association between behavioral, physiological, and EEG responses within CD and MCD treatments. The data demonstrate that pentobarbital sodium induced a rapid death with minimal behavioral and physiological responses regardless of strain of hens. In comparison, use of CD and MCD resulted in a slow onset of brain death in hens.

Dataset Information

Evaluating Intrahepatic and Intraperitoneal Sodium Pentobarbital or Ethanol for Mouse Euthanasia.

Publications

Evaluating Intrahepatic and Intraperitoneal Sodium Pentobarbital or Ethanol for Mouse Euthanasia.

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