Thyroid diseases, associated with either increased or decreased concentrations of circulating thyroid hormones, are prevalent in both human and veterinary populations. Hypothyroidism is a differential diagnosis for many medical problems as the disease presents with nonspecific clinical signs that can include lethargy, weight gain, cold intolerance, and dermatologic manifestations such as alopecia. Alopecia is a frequently reported problem in captive nonhuman primates (NHP), and hypothyroidism is considered to be a differential diagnosis. However, thyroid function test results in NHP using total T4 (TT4) and free T4 (FT4) assays are difficult to interpret without accurate reference intervals (RI) for comparison. As a consequence, hypothyroidism may be underdiagnosed in these species. The objective of this study was to establish RI for TT4 and FT4 in healthy populations of cynomolgus macaques ( n = 133; age range 2.6 to 24.7 y) and rhesus macaques ( n = 172; age range 0.8 to 31.0 y). Serum samples were collected across a 14-y period during routine anesthetic events in clinically healthy animals, and TT4 and FT4 concentrations were measured using commercially available immunoassays. The RI established for TT4 and FT4 were 5.1 to 14.9 ug/dL and 0.48 to 1.17 ng/dL for cynomolgus macaques, and 3.9 to 14.7 ug/dL and 0.36 to 1.12 ng/dL for rhesus macaques. Significant differences in thyroid hormone concentrations were found between Indian and Chinese origin rhesus, and between Mauritian and other origin cynomolgus. In addition, juvenile and subadult rhesus exhibited significantly higher FT4 and TT4 concentrations than did older animals. Individual RI were established for subgroups with adequately different thyroid hormone concentrations. These results will allow a more thorough diagnostic evaluation of cynomolgus and rhesus macaques with clinical signs consistent with thyroid disease and will ultimately be a refinement in NHP medicine.

Vitrification negatively affects the mitochondrial membrane potential (ΔΨm) in oocytes while also leading to increased reactive oxygen species (ROS), ATP depletion and induction of apoptosis in oocytes. Mitoquinone (MitoQ) is an antioxidant that protects mitochondrial membrane integrity from ROS. This study examined the effect of adding MitoQ to vitrification medium on mitochondrial function and embryo development in vitrified oocytes. Metaphase II (MII) stage oocytes were collected from NMRI mouse ovaries and preincubated for 20 min in a medium containing 0.02 μM of MitoQ. Next, oocytes were vitrified in medium supplemented with 0.02μM of MitoQ (treatment group). The control group was processed in the same way but without exposure to MitoQ. After warming, oocyte survival rate, ΔΨm, cytoplasmic ROS and glutathione (GSH) levels and gene expression levels (Bcl2, BAX, and caspase3) were measured. In addition, the vitrified oocytes were fertilized in-vitro to assess developmental competence. The results showed that MitoQ improved survival and ΔΨm in treated vitrified oocytes. Treated oocytes showed lower ROS levels and higher GSH levels than did the control group. Furthermore, mRNA expression of the Bax/ Bcl2 ratio and caspase3 were significantly lower in treated oocytes. These findings indicate that medium supplementation with 0.02 μM of MitoQ during vitrification can improve oocyte survival and developmental competency in mouse oocytes.

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Understanding how differences in animal husbandry practices affect the reproducibility of research results is critical. We sought to understand how different beddings might influence dietary obesity studies. We compared the effects of paper and corncob bedding on weight gain, metabolism, and gut microbiome (GM) of mice fed a high-fat diet (HFD) or a normal diet (ND) and evaluated effects on fecal and cecal microbiomes collected from these cohorts after euthanasia. Male C57BL/6J mice at 5 wk age were allowed to acclimate to the facility and the assigned bedding for one week before being placed on HFD or remaining on the ND for 12 wk. Fecal pellets and cecal samples were collected and frozen for batched 16S sequencing. Mice had similar body weight, visceral gonadal white adipose tissue (GWAT), subcutaneous inguinal white adipose tissue (IWAT), liver and spleen weights and metabolic changes regardless of the bedding type. Baseline microbiota differences were detected one week after bedding assignment. After 12 wk, the GM showed significant differences depending on both bedding and diet. The effects of the bedding were not significantly different between endpoint fecal and cecal GM, despite the inherent differences in microbiota in fecal and cecal samples. A correlation was detected between diet and the relative abundance of Bacteroidetes and Verrucomicrobia: Akkermansia. In conclusion, this study demonstrates the importance of considering bedding type when performing dietary experiments.

Compassion fatigue (CF) has been described in various "caring professions," particularly the human medical field. Recently, CF has been identified as a concern in animal care professions, specifically veterinary medicine. Despite the perception that veterinary personnel in animal research are at increased risk of CF, few studies have assessed CF in this population. The current cross-sectional study aimed to describe the prevalence of both CF and compassion satisfaction (CS) among active-duty veterinary personnel in Department of Defense animal research environments, using the Professional Quality of Life Scale (ProQOL). The ProQOL measures burnout and secondary traumatic stress as representative of compassion fatigue, while also measuring the inverse of CF, or CS. The current study set out to identify factors associated with both CF and CS. Validated scales of measurement were used to assess the frequency of exposures and outcomes of interest, while associations were analyzed using linear regression models. The study found that most survey respondents reported high levels of CS and low levels of BO and STS. Factors associated with higher levels of CF and lower CS included working with NHPs, difficulty working with primary investigators, loneliness, and euthanasia distress. These findings can inform future studies of CF in animal research environments and bolster initiatives to reduce occupational stress by the preventing and mitigating CF.

Reuse of disposable personal protective equipment is traditionally discouraged, yet in times of heightened medical applications such as the SARS CoV-2 pandemic, it can be difficult to obtain. In this article we examine the reuse of disposable gowns with respect to still providing personnel protection. XR7, a fluorescent powder, was used to track contamination of gowns after manipulation of rodent cages. Mouse cages were treated with XR7 prior to manipulations. Disposable gowns were labeled for single person use and hung in common procedure spaces within the vivarium between usages. A simulated rack change of 140 cages was completed using XR7-treated cages. One individual changed all cages with a break occurring after the first 70 cages, requiring the gown to be removed and reused once. To simulate research activities, 5 individuals accessed 3 XR7-treated cages daily for 5 d. Each mouse in the XR7-treated cages was manipulated at least once before returning cages to the housing room. Disposable gowns were reused 5 times per individual. Gowns, gloves, clothing, bare arms, and hands were scanned for fluorescence before and after removing PPE. Fluorescence was localized to gloves and gown sleeves in closest contact with animals and caging. No fluorescence was detected on underlying clothing, or bare arms and hands after removing PPE. Fluorescence was not detected in procedure spaces where gowns were hung. The lack of fluorescence on personnel or surfaces indicate that gowns can be reused 1 time for routine husbandry tasks and up to 5 times for research personnel. A method for decontamination of used gowns using Vaporized Hydrogen Peroxide (VHP) was also validated for use in areas where animals are considered high risk such as quarantine, or for fragile immunocompromised rodent colonies.

Studies published in 1994 and 2000 established a temperature range of 143–180 °F for effective cage sanitization in animal facilities. These 2 studies were, respectively, theoretical and based on experiments using hot water to sanitize bacteria-coated test tubes. However, such experimental methods may not capture the practical advantages of modern washing technology or account for the routine use of detergent in cage wash. Moreover, these methods may not translate to the challenges of removing adhered debris and animal waste from the surfaces being sanitized. A sample of highly soiled cage bottoms, half of which were autoclaved with bedding to create challenging cleaning conditions, were processed at 6 combinations of wash and rinse cycles with 125 °F, 140 °F, and 180 °F water with detergent. All cycles were equipped with a data logging device to independently verify temperatures. After washing, cages underwent visual inspection and microbial sampling consisting of organic material detection using ATP detection and Replicate Organism Detection and Counting (RODAC) plates. Cages with any amount of visible debris failed inspection, as did cages that exceeded institutional sanitization thresholds. Results indicate that wash and rinse temperatures of 140 °F for a programmed wash duration of 450 s and rinse of 50 s effectively clean and disinfect both highly soiled and autoclaved cages. Accounting for both steam and electrical energy, these parameters result in an annual savings of $21,867.08 per washer on an equivalent run basis using the current institutional standard of 180 °F.

Pathogenesis of viruses or other agents that are infectious to humans is frequently studied in vivo using natural or genetically modified animals. Depending on the risk group of the pathogen, the majority of such experimental studies are performed at least under biosafety level 2 (BSL-2) conditions. Biosafety considerations are therefore critical at all steps of research involving potentially infectious pathogens. Inactivation of pathogens studied using in vitro experiments is usually performed using moist heat sterilization. However, few standardized and validated protocols are currently available for the thermal inactivation of carcasses from laboratory animals infected with such human pathogens. To comply with laboratory biologic safety rules and requirements imposed by regulatory authorities, documentation of appropriate inactivation conditions or use of a validated procedure according to national or international standards is critical. In the current study, we evaluated inactivation protocols in a standard laboratory autoclave for carcasses of either frozen mice or recently terminated rabbits, which were placed inside autoclave bags with bedding material in stainless steel containers. Temperature sensors were placed into different tissues of the carcasses to continuously record temperature in situ and in real-time, and a reference sensor was placed in the autoclave. To achieve pathogen inactivation, autoclaving protocols had to be optimized for both species. Frozen mice required 2 different fractionated prevacuum stages, whereas recently terminated rabbits required 3 different fractionated prevacuum stages. This study provides a template for an evaluation procedure to safely and effectively inactivate mice and rabbits infected with risk group 2 to 4 pathogens.

A novel buprenorphine (BUP) extended-release formulation (BUP-XR) produced as a lipid-encapsulated, low viscosity BUP suspension for SC injection to control pain was evaluated for pharmacokinetics and safety in Sprague–Dawley rats given either 0.65 mg/kg (low dose) or 1.30 mg/kg (high dose). The 2 dosage groups each contained 6 male and 6 female rats to determine whether BUP-XR behaved differently in male or female animals. Blood samples were obtained from each animal before BUP-XR administration and at 6, 24, 48, 72, 96, and 168 h after administration. For necropsy and injection-site histopathology evaluation, 3 animals of each sex from each test group were euthanized on day 8, with the remaining animals euthanized on day 15. Mean plasma BUP concentration peaked from 6 to 24 h in all test groups, then declined in a linear fashion. Quantifiable plasma BUP was measured in all male rats at all time points except for one low dose group sample taken at 168 h. Female rats had quantifiable plasma BUP at all time points except for 1 low dose group sample at 72 and 96 h, and 2 low dose group samples at 168 h. The low dose groups, whether male or female, had lower mean plasma BUP levels at all time points as compared with their high dose counterparts, and female rats had lower mean plasma BUP levels than male rats at all time points. Results indicate that a single BUP-XR dose at either dose concentration can reliably provide plasma levels of BUP reported in the literature to be therapeutically relevant for up to 72 h, although lower plasma BUP levels can be anticipated in female rats compared with male counterparts. Mild to moderate injection-site granulomatous inflammation was observed in 6 of 12 rats in the low dose group and 7 of 12 in the high dose group. This reaction is characteristic of lipid material designed to persist in situ.

Carprofen, a nonsteroidal antiinflammatory drug, is a commonly used analgesic for laboratory animals. The manufacturer labeling indicates the stock bottle may be stored and used for up to 28 d under refrigeration. However, institutional guidelines may vary in how long diluted carprofen solutions can be stored before they must be discarded. When administered to laboratory rodents, small volumes of the stock solution are diluted to provide accurate dosing and ease of administration. Because carprofen is formulated for use in companion animals (for example, dogs) and comes in larger volume multidose vials, the majority of carprofen at our institution is discarded before it can be used. In this study, we evaluated the amount of target ingredient present (strength), sterility, and endotoxin levels of both stock and diluted carprofen when stored in a variety of containers and at multiple temperature settings for up to 180 d, mimicking facets of typical use in laboratory animal research and medicine. We demonstrated that when refrigerated and stored in sterile vials, stock and diluted carprofen can be kept and used for up to 180 d while retaining strength and sterility. For short-term use, diluted carprofen can also be stored for up to 60 d at room temperature in conical tubes. These results will help establish scientifically justified storage conditions for carprofen to ensure that these agents remain appropriately potent and sterile for therapeutic use in laboratory animals.

Trichuris spp. are common helminths in NHP, and benzimidazoles and avermectins have both been used to treat these intestinal parasites. The current study compared the efficacy of fenbendazole and ivermectin against natural infection of Trichuris spp. in African green monkeys (Chlorocebus sabaeus). Anthelmintic-naive animals (n = 65) were randomly assigned to 4 groups: an untreated control group, and 3 groups treated with either fenbendazole, ivermectin, or both compounds. Fecal samples were collected before treatment and on days 7, 14, 28, and 60 after treatment, and fecal egg counts (FEC) were determined by using fecal flotation. The mean percentages of FEC reduction at day 60 were 100%, 86%, and 100% for treatment with fenbendazole, ivermectin, and both compounds, respectively. Analyzing the time series of FEC by using a Bayesian generalized linear model showed no significant difference in the proportional reduction in FEC among the 3 treatment groups, although all FEC from treated groups were significantly lower than the FEC of the control group. In contrast, the probability of shedding was highest in the ivermectin group and the lowest in the animals treated with both compounds. The probability of shedding differed significantly between the fenbendazole and ivermectin groups and between the ivermectin and combined-treatment groups. In conclusion, both fenbendazole and ivermectin are effective anthelmintics in treating Trichuris spp. infection in African green monkeys. All treatment groups showed significant reductions in FEC when compared with baseline counts and control animals; however, fenbendazole may be more effective than ivermectin when used solely or in combination with other anthelmintic treatments.

Alopecia occurs frequently in captive populations of nonhuman primates. Because multiple factors can play a role in alopecia, a better understanding of its etiology will help identify potential welfare concerns. The purpose of this study was to investigate risk factors for alopecia in a breeding colony of baboons with a focus on pregnancy and age. Alopecia was scored on a scale of 0 (no alopecia) to 5 (severe alopecia) in 253 female baboons during routine physicals. The subjects ranged in age from 4 to 23 y (Mean = 9.6) and were categorized as pregnant (n = 83), nursing (n = 60) or control (n = 110). Resulting alopecia scores were combined into 2 categories (mild = 0 or 1; moderate = 2 or 3); no animals scored a 4 or 5. Significantly more pregnant females had moderate alopecia than did control females. There was no effect of age on alopecia. An unexpected outcome was that among nursing females, more of those with female infants had moderate alopecia than did those with male infants. The impact of the infant's sex on alopecia may be due to sex differences in maternal contact or maternal investment. This information adds to our understanding of alopecia risk factors in captive nonhuman primates.