The goal of this study was to develop a method for ultrasound-guided percutaneous intraovarian injection in Wistar rats. Intraovarian administration of chemicals or needle aspiration of the ovary has been undertaken in some species, including humans, equines, and bovines. In rodents, which are widely used in scientific research, a technique for intraovarian injection without surgical exposure of the organ has not been described. The current study standardized the procedure of ovarian percutaneous injection of 0.9% sodium chloride guided by ultrasound in rats. The ovaries were measured by ultrasound imaging before and immediately after injection and showed a significant increase in ovarian length but not width. No clinical abnormalities were detected within 15 d after injection. These findings indicate that the steps of ultrasound localization of the organ, digital restraint, and correct needle insertion achieved successful intraovarian administration of saline without invasive surgery. These results document the feasibility of ultrasound-guided intraovarian percutaneous injection in rats and may be useful for future research on female reproduction and chemical sterilization.

The Syrian hamster (Mesocricetus auratus) is a solitary and naturally territorial animal, with female hamsters being more aggressive than males. This behavior makes handling difficult because they are usually housed in groups, which can lead to aggressive behavior. The objective of this study was to refine the management of Syrian hamsters in order to minimize aggressiveness, reduce the animal injuries, and lessen the risk of accidents among laboratory animal technicians due to the hamster aggression during handling. The experiment was conducted at the Center for Animals Experimentation, Oswaldo Cruz Institute. Four groups of hamsters were observed by video recording: group 1 (group-housed males, 6 to 8 wk of age), group 2 (group-housed females 6 to 8 wk of age), group 3 (group-housed female, 3 to 4 wk of age), and group 4 (individually housed females, 6 to 8 wk of age). Group 1 animals were less aggressive and agitated both during housing and during handling by the animal technician as compared with groups 2 and 3. Groups 2 and 3 showed greater agitation and aggression. Marked reduction in the level of aggressiveness and agitation was observed in group 4 as compared with all other groups evaluated during handling by the animal technician. Male hamsters housed in groups of 4 and females housed individually have reduced risks of accident during handling, thereby averting distress and consequent physiologic alterations. Avoiding these risks is essential to obtaining reliable experimental results.

Pathogen transmission into zebrafish colonies is controlled through vigilant biosecurity practices. One such practice is embryo surface disinfection, which often uses sodium hypochlorite. However, if sodium hypochlorite is used at an inappropriate pH, concentration, or exposure time, zebrafish embryos can experience significant mortality and morbidity. Reagent-grade sodium hypochlorite is often used for embryo surface disinfection because commercial-grade sodium hypochlorite has additional ingredients that may have deleterious effects on the embryo. In addition, chlorine dioxide and the combination of sodium chloride and potassium peroxymonosulfate (SCPP) are effective equipment disinfectants; however, the effects of these chemical agents on zebrafish embryos during surface disinfection are unknown. In this study, we exposed strain 5D zebrafish embryos (ages, 6 and 24 h postfertilization) to 4 chlorine-containing agents (reagent-grade sodium hypochlorite [bleach], commercial-grade sodium hypochlorite [bleach], SCPP, and chlorine dioxide) at either 50- or 100- ppm for 5 or 10 min. All groups were evaluated at 5 d postfertilization for survival, hatching rate, and morphologic defect rate. The experimental group with the highest survival and hatching rates and the lowest morphologic defect rate was the 24-h postfertilization embryos exposed to 50 ppm SCPP for 5 min. The survival, hatching rate, and defect rate did not differ significantly among age-matched controls; however, the hatching rate after exposure to 50 ppm SCPP was significantly higher than that of embryos exposed to 50 ppm reagent-grade sodium hypochlorite for 5 min (100% compared with 23% respectively). SCPP solution may provide an alternative surface disinfectant for zebrafish embryos because it maximizes survival and hatching rates and minimizes morphologic defect rates. However, in vivo efficacy against common zebrafish pathogens requires further testing. Use of chlorine dioxide at 50 ppm or greater is not recommended for zebrafish embryo surface disinfection due to significant mortality among 6 and 24 h postfertilization embryos.

Water regulation is a procedure that allows animals to consume water volumes equivalent to ad libitum access, but access is limited to specific time intervals (that is, water is not available outside of the designated access periods). Despite the relatively common use of water regulation in research, the implementation method is rarely detailed, stating only that water was available in the animal's home cage at specific times. For planned toxicologic assessments, we placed rats (n = 510) and ferrets (n = 16) on water regulation using both automated and manual methods. In testing our systems, we defined "successful implementation" as maintenance of appropriate weight gain and health status. An automated system that controlled water access to an entire rat rack was successful for most rats, but several rats failed to consume enough water even after 2 wk of experience. Manual methods of water regulation were successful in rats by either moving the cage to prevent access to the drinking valve or by placing/removing water bottles. An automated system that controlled water access from water bottles was implemented for ferrets and was maintained for up to 30 wk. Retrospective comparison of body weights to standard growth curves for both species showed that all animals grew normally despite water regulation. Differences in the systems and some species considerations provide insights into the key elements necessary for successful water regulation in rats and ferrets.

Cage washing is a key process of the biosecurity program in rodent facilities. For the current study, we developed systems (i. e., magnet attachments, quantitative biologic indicators (Q-BI), and measurement of thermal disinfection at equipment level) to assess the microbial decontamination achieved by a rodent equipment washer with and without thermal disinfection. 99% of the magnets remained in position to hold Q-BI and temperature probes inside cages, water bottles or at equipment level across a cabinet washer chamber with loads dedicated to either housing or drinking devices. Various types of Q-BI for Bacillus atrophaeus, Enterococcus hirae and minute virus of mice were tested. To simulate potential interference from biologic material and animal waste during cage processing, Q-BI contained test soil: bovine serum albumin with or without feces. As a quantitative indicator of microbial decontamination, the reduction factor was calculated by comparing microbial load of processed Q-BI with unprocessed controls. We detected variation between Q-BI types and assessed the washer's ability to reduce microbial load on equipment. Reduction factor results were consistent with the Q-BI type and showed that the washing and thermal disinfection cycle could reduce loads of vegetative bacteria, virus and spore by 5 log₁₀ CFU/TCID₅₀ and beyond. Thermal disinfection was monitored with temperature probes linked to data loggers recording live. We measured the period of exposure to temperatures above 82.2 °C, to calculate A₀, the theoretical indicator for microbial lethality by thermal disinfection, and to assess whether the cabinet washer could pass the minimum quality standard of A₀ = 600. Temperature curves showed an A₀ > 1000 consistently across all processed equipment during thermal disinfection. These data suggest that, when sterilization is not required, a cabinet washer with thermal disinfection could replace an autoclave and reduce environmental and financial waste.

Due to their reduced frequency of dosing and ease of availability, NSAIDs are generally preferred over opioids for rodent analgesia. We evaluated the efficacy of the highly COX2-selective NSAID firocoxib as compared with meloxicam and buprenorphine for reducing allodynia and hyperalgesia in rats in a plantar incision model of surgical pain. After a preliminary pharmacokinetic study using firocoxib, Sprague–Dawley rats (n = 12 per group, 6 of each sex) were divided into 6 groups: no surgery (anesthesia only), saline (surgery but no analgesia), buprenorphine (0.05 mg/kg SC every 8 h), meloxicam (2 mg/kg SC every 24 h), and 2 dosages of firocoxib (10 and 20 mg/kg SC every 24 h). The nociception assays were performed by using von Frey and Hargreaves methodology to test mechanical allodynia and thermal hyperalgesia. These assays were performed at 24 h before and at 20, 28, 44, and 52 h after start of surgery. None of the analgesics used in this study produced significantly different responses in allodynia or hyperalgesia from those of saline-treated rats. In the Hargreaves assay, female saline-treated rats experienced significantly greater hyperalgesia than did males. These findings add to a growing body of literature suggesting that commonly used dosages of analgesics may not provide sufficient analgesia in rats experiencing incisional pain.

In biomedical research, rabbits are commonly sedated to facilitate a variety of procedures. Developing a sedation assessment scale enables standardization of levels of sedation and comparisons of sedation protocols, and may help in predicting sedation level requirements for different procedures. The goal of this study was to develop a rabbit sedation assessment scale using a psychometric approach. We hypothesized that the sedation scale would have construct validity, good internal consistency, and reliability. In a prospective, randomized, blinded study design, 15 (8 females, 7 males) healthy 1-y-old New Zealand white rabbits received 3 intramuscular treatments: midazolam (0.5 mg/kg; n = 6); midazolam (1.5 mg/kg)–ketamine (5 mg/kg; n = 7); and alfaxalone (4 mg/kg)–dexmedetomidine (0.1 mg/kg)–midazolam (0.2 mg/kg; n = 3). One rabbit received 2 treatments. A sedation scale was developed by using psychometric methods, with assessment performed by 6 independent raters who were blind to treatment. Final sedation scale items included posture, palpebral reflex, orbital tightening, lateral recumbency, loss of righting reflex, supraglottic airway device placement, toe pinch, and general appearance. The scale showed construct validity, good to very good interrater reliability for individual items (6 raters; intraclass correlation coefficient, 0.671 to 0.940), very good intrarater reliability (5 raters; intraclass correlation coefficient, 0.951 to 0.987), and excellent internal consistency (Cronbach α, 0.947). The sedation scale performed well under the conditions tested, suggesting that it can be applied in a wider range of settings (different populations, raters, sedation protocols).

Cephalopods are important in biologic and biomedical research, yet relatively little objective information is available to guide researchers and veterinarians regarding the best methods for anesthetizing these animals for various experimental procedures. Recent studies demonstrate that ethyl alcohol and magnesium chloride are effective at depressing efferent and afferent neural signals in some tropical cephalopod species when measured via the pallial nerve. Here we used similar methods to test 2 temperate species (Octopus bimaculoides and Sepia officinalis) and demonstrate that (1) ethyl alcohol and magnesium chloride were effective at reversibly depressing evoked activity in the pallial nerve, (2) ethyl alcohol generally had shorter induction and recovery times compared with magnesium chloride, (3) both agents were associated with a latency between the behavioral and neural effects, and it was longer with magnesium chloride, and (4) senescent animals generally had longer induction or recovery times than young animals. Both agents successfully anesthetized both life stages; however, our data show that assessing anesthesia based solely on behavior may lead to premature commencement of invasive procedures. We conclude that temperate cephalopods can be humanely, effectively, and completely anesthetized by using these 2 agents and that the loss of neural signal we show here is consistent with true anesthesia and not merely paralysis. This relatively simple, nondestructive nerve recording technique can be applied to the study of other prospective anesthetic agents in cephalopods.

Although buprenorphine is the most frequently used opioid analgesic in common marmosets (Callithrix jacchus), there is limited information in the literature supporting current dosing regimens used for this species. The purpose of this study was to determine the pharmacokinetic profiles of single-dose buprenorphine HCl administered intramuscularly (IM) at 0.01 mg/kg in 6 adult marmosets (1.8 to 12.8 y old; 2 males, 4 females) and subcutaneously (SQ) at 0.01 mg/kg in 6 adult marmo- sets (2.3-4.4 y old; 3 males, 3 females) by mass spectrometry. Blood was collected at multiple time points from 0.25 to 24 h from unsedated animals following a hybrid sparse-serial sampling design. The maximal observed plasma concentration of buprenorphine (C_max ) administered IM (2.57 ± 0.95 ng/mL) was significantly higher than administered SQ (1.47 ± 0.61 ng/mL). However, the time to C_max (T_max) was not statistically different between routes (17.4 ± 6 min for IM and 19.8 ± 7.8 min for SQ). The time of the last quantifiable concentration of buprenorphine was 5 ± 1.67 h for IM compared with 6.33 ± 1.51 h for SQ, which was not statistically different. The mean buprenorphine plasma concentration-time curves were used to propose a dosing frequency of 4 to 6 h for buprenorphine at 0.01 mg/kg IM or SQ based on a theoretical therapeutic plasma concentration threshold of 0.1 ng/mL. Based on the mean pharmacokinetic parameters and plasma-concentration time curves, both IM and SQ routes of buprenorphine at this dose provide a rapid increase in the plasma concentration of buprenorphine above the therapeutic threshold, and may be more effective for acute rather than long-lasting analgesia. Further studies are needed to examine repeated dosing regimens and the efficacy of buprenorphine in common marmosets.

Ophthalmic examination (OE) is a common part of preclinical studies. Pupillary dilation to facilitate OE may affect results of other planned testing. The purpose of this study was to determine the duration of mydriasis produced by commercially available 0.5% and 1% tropicamide in ophthalmologically normal albino rats. Twelve female Sprague–Dawley rats were used. A single drop of 1% tropicamide was applied to one eye of each rat. A single drop of balanced salt solution (BSS) was applied to the contralateral eye. Measurements of pupillary diameter (PD) were obtained using a digital caliper at 0, 20, 40, 60, 120, 180, 240, 300, and 360 min after application. After a 3-wk washout period, the procedure was repeated using 0.5% tropicamide. Pupillary dilation sufficient to allow posterior segment evaluation was achieved with 0.5% and 1% tropicamide. Maximum PD after treatment with 0.5% tropicamide was 4.17 ± 0.22 mm at 40 min; maximum PD after treatment with 1% tropicamide was not significantly different (4.28 mm at both 20 and 40 min (±0.43 mm and 0.23 mm, respectively)). Mean PD remained above 3.5 mm in treated eyes for 60 min. In eyes treated with 0.5% tropicamide, mean PD was significantly different from baseline mean PD for that eye up to 300 min. In eyes treated with 1% tropicamide, mean PD was significantly different than baseline mean PD for that eye at all timepoints. Both concentrations of tropicamide produced a transient mild to moderate mydriasis in the contralateral eye. Duration of action is at least 5 h for 0.5% tropicamide and 6 h for 1% tropicamide. Results of this study support use of 0.5% tropicamide for OE in albino rats, with administration performed no more than 60 min prior to examination.

The African clawed frog, Xenopus laevis, is a widely used model for biomedical research. X. laevis could be more useful as a model with a better method for collection and analysis of its blood and serum. However, blood collection in X. laevis can be challenging due to their small size, lack of peripheral vascular access, and species-specific hematology variables. The goal of this study was to compare cardiocentesis, the current gold standard terminal blood collection method, with a leg amputation technique. Blood samples were collected from 24 laboratory-reared X. laevis, randomized to either the cardiocentesis or leg amputation method, with 6 males and 6 females in each group. Hematology and serum biochemistry were also conducted to identify any lymph contamination in the samples. The leg amputation method produced significantly higher blood volumes in shorter times and showed no significant differences in clinical pathology parameters as compared with cardiocentesis. These results indicate that blood collection by leg amputation may be a valuable approach for increasing the utility of an already valuable biomedical research model.

Tinidazole is a second-generation nitroimidazole compound that is used as an antimicrobial to treat anaerobic bacterial and protozoal infections in humans and, less frequently, in veterinary medicine. However, metronidazole, another secondgeneration nitroimidazole, is more commonly used. Nonetheless, tinidazole has proven to be a superior therapy for parasitic infections in humans, particularly in the treatment of giardiasis. Furthermore, in chinchillas, metronidazole has been shown to cause a clinically relevant reduction in food intake after oral administration at published dosages. This study's objective was to evaluate the effect of orally administered tinidazole on food intake in healthy chinchillas. In 2 randomized, placebocontrolled, blinded, crossover studies, tinidazole was evaluated at 2 single high doses (100 mg/kg and 200 mg/kg PO, n = 9) and a repeated dosing schedule at a lower dose (20 mg/kg PO q12h for 10 doses, n = 12). Food intake was measured over 24-h periods before and after drug administration. The single-dose treatment groups both displayed significantly reduced food intake (200 mg/kg: –26 ± 21%; 100 mg/kg: –9 ± 21%, P < 0.01) as compared with the control group during the first 24 h after drug administration. Food intake returned to pretreatment values within 4-5 d. Repeated administration at 20 mg/kg q12h was not associated with any significant changes in food intake. No other adverse effects were noted during this study. Tinidazole administration at single higher doses resulted in an acute self-limiting reduction in food intake. In comparison, repeated administration of lower doses (20 mg/kg PO q12h) had no significant effects on food intake in healthy chinchillas. Therefore, tinidazole may be a more suitable drug for treating Giardia and anaerobic bacterial infections in this species than the more commonly used metronidazole.