Search

Despite the major use of mice in biomedical research, little information is available with regard to identifying their postmortem changes and using that information to determine the postmortem interval (PMI), defined as the time after death. Both PMI and environmental conditions influence decomposition (autolysis and putrefaction) and other postmortem changes. Severe decomposition compromises lesion interpretation and disease detection and wastes limited pathology resources. The goal of this study was to assess postmortem changes in mice in room temperature cage conditions and under refrigeration at 4 °C to develop gross criteria for the potential value of further gross and histologic evaluation. We used 108 experimentally naïve C57BL/6 mice that were humanely euthanized and then allocated them into 2 experimental groups for evaluation of postmortem change: room temperature (20 to 22 °C) or refrigeration (4 °C). PMI assessments, including gross changes and histologic scoring, were performed at hours 0, 4, 8, and 12 and on days 1 to 14. Factors such as temperature, humidity, ammonia in the cage, and weight change were also documented. Our data indicates that carcasses held at room temperature decomposed faster than refrigerated carcasses. For most tissues, decomposition was evident by 12 h at room temperature as compared with 5 d under refrigeration. At room temperature, gross changes were present by day 2 as compared with day 7 under refrigeration. Mice at room temperature lost 0.78% of their baseline body weight per day as compared with 0.06% for refrigerated mice (95% CI for difference 0.67% to 0.76%, P < 0.0005). This study supports the consideration of temperature and PMI as important factors affecting the suitability of postmortem tissues for gross and histologic evaluation and indicates that storage of carcasses under refrigeration will significantly slow autolysis.

Isoflurane has been characterized as a distressing agent for rodents, causing both physiologic and behavioral effects. Using a "darkened home cage" has been recommended during CO₂ administration for rodent euthanasia; this is arguably a similar animal experience to anesthetic induction with isoflurane. Based on the premise that rodents perceive red light as darkness via the primary optic tract, we compared physiologic and behavioral markers of stress in 2 inbred strains of mice (C57BL/6J and BALB/cJ) anesthetized with isoflurane in either a red-tinted (dark) induction chamber or a traditional translucent induction chamber. Physiologic stress was assessed based on plasma levels of norepinephrine, epinephrine, and corticosterone. Stress-related behaviors (rearing, face wiping, and jumping) were recorded on video and scored from initiation of induction to loss of consciousness. No significant correlations were found between chamber type and physiologic stress hormones. As compared with the translucent chamber, stress-related behaviors were more frequent in the red-tinted chamber, including: 1) significantly higher rearing frequencies in BALB/cJ mice; 2) higher behavioral stress scores in BALB/cJ and male C57BL/6J mice; and 3) more face wiping behavior when considering all mice combined. These findings suggest that mice do not experience significant alleviation of physiologic indices of stress when anesthetized in a red-tinted induction chamber. Furthermore, isoflurane induction in the red-tinted chamber appeared to increase the expression of stress-related behaviors, particularly in BALB/cJ mice. Based on our findings and a growing body of literature on the unintended effects of red light, we do not recommend using red-tinted chambers for induction of anesthesia in mice.

One strategy commonly employed for rodent surgeries is a "tips-only" surgical technique, which restricts the surgeon to using only the sterile working ends of the surgical instruments to manipulate the surgical field and sterilizes instrument tips with a hot bead sterilizer between consecutive rodents. Despite the common use of the "tips-only" technique, research is lacking on the number of sequential surgeries for which the same set of hot bead-sterilized instruments can be used before introducing bacterial contamination. We performed serial mouse surgeries using the "tips-only" technique under 3 different conditions (aseptic, fur contamination, or cecal contamination) and assessed aerobic bacterial growth before and after each round of hot bead sterilization. Instrument tips showed an increasing probability of contamination of at least one instrument in a series of consecutive surgeries. The probability that all surgical instrument tips in the series were sterile after hot bead sterilization fell by 4% for each surgery involving inadvertent or fur contamination and by 11.5% for each surgery with contamination for all surgical types combined (including entering the gastrointestinal tract). Based on our results, hot bead sterilization is not adequate for surgeries associated with gross contamination. Under our experimental conditions and assuming independence of outcomes between consecutive surgeries, up to 5 surgeries associated with minor or inadvertent contamination could be performed in series with a probability higher than 80% that all instrument tips were sterile for all surgeries. A case-by-case risk assessment should be conducted to derive institutional guidelines for the maximal number of surgeries that can be performed in sequence using the "tips-only" technique with hot bead sterilization of the same set of surgical instruments between surgeries. Full sterilization of instruments after every surgery provides the greatest confidence in maintaining sterility.

The Northern greater galago ( Otolemur garnettii ) is a prosimian primate most commonly used to study the evolutionary development of vision and somatosensation. This study aimed to investigate the efficacy and cardiopulmonary effects of 3 sedation protocols commonly used in other primate species: 1) alfaxalone (Alf; 8 mg/kg IM) 2) ketamine alone (Ket; 20 mg/kg IM) and 3) ketamine + dexmedetomidine (Ket+Dex; 4 mg/kg + 25 μg/kg IM) with reversal (atipamezole; 250 μg/kg IM). A total of 34 animals were evaluated, including 11 juveniles and 23 adults. Cardiopulmonary parameters such as indirect blood pressure, heart rate, respiratory rate, and SpO₂ were measured, and blood was collected for blood gas analysis and a chemistry panel. To examine the efficacy of each sedation protocol, induction time, immobilization time, and recovery time were recorded. Subjective measures of quality and efficacy included quality of induction, pedal withdrawal reflex, palpebral reflex, muscle tension, rectal temperature, and quality of recovery. All 3 protocols successfully immobilized the animals and all animals recovered from sedation. Heart rates were highest among the Ket group and the lowest for the Ket+Dex group. On average, the Alf group was immobilized for twice as long as either the Ket or Ket+Dex groups. The Ket+Dex group had the fastest average recovery time and subjectively had the best quality of recovery. Based on these results, Ket+Dex is recommended over Alf or Ket alone for brief sedation of healthy galagos.

Invertebrates are often overlooked as laboratory animals, yet they are commonly used in toxicology, developmental, cellular and molecular biology, and radiation studies with euthanasia as an endpoint. Little is known regarding appropriate euthanasia methods for invertebrate species, particularly for Artemia . Here, we evaluated the AVMA-recommended 2-step method of euthanasia in brine shrimp ( Artemia franciscana ). Artemia were exposed first to anesthetic solutions of 60% alcohol, 2.5 mg/L eugenol, or 4 g/L tricaine methanesulfonate (TMS) and then were transferred to euthanasia solutions of 70% alcohol, 95% alcohol, or 10% neutral buffered formalin. We examined time to anesthesia, behavioral response to anesthesia, anesthesia recovery, and time to euthanasia. Our results show that 2.5 mg/L eugenol and 4 g/L TMS inconsistently achieved anesthesia. Although 60% alcohol produced anesthesia, the time to anesthesia varied among replicate groups, and exposure resulted in an increase in abnormal behavior. We therefore do not recommend any of the tested anesthetic solutions for use in Artemia. Although all 3 euthanasia solutions were effective, more research is needed to provide recommendations regarding euthanasia for this species.

Sexually mature zebrafish were housed as single male-female pairs with or without plastic vegetation for 1, 5, or 10 d for comparison of whole-body cortisol measured by radioimmunoassay. Individually housed male zebrafish were used as controls. In the fish that were pair-housed without vegetation (NVeg), one animal died in 5 of 24 pairs, and one animal was alive but wounded in an additional pair. No deaths or wounds occurred in the fish that were pair-housed with vegetation (Veg). Cortisol levels did not differ between the treatment groups on day 1. On day 5, cortisol values were higher in the Veg group than in the individually housed fish (P < 0.0005) and the NVeg fish (P = 0.004). On day 10, the relationships were inversed: cortisol levels had risen in the individually housed and NVeg groups and had fallen to baseline levels in the Veg group. Cortisol values on day 10 were lower in the Veg group than in the individually housed (P = 0.004) and NVeg (P = 0.05) groups. Cortisol levels in individually housed male zebrafish increased over time. Although this study did not demonstrate a reduction in cortisol levels associated with providing vegetation, this enrichment prevented injury and death from fighting. These findings show how commonly used housing situations may affect the wellbeing of laboratory zebrafish.