In pharmaceutical development, weight loss is occasionally observed in monkeys during non-clinical toxicity studies and can be difficult to differentiate from drug effects. This study retrospectively analyzed data from control group monkeys without drug treatment to investigate the incidence of weight loss and its physiological and pathological characteristics. We also investigated potential improvements through enhanced animal welfare. In the 4- and 13-week toxicity studies conducted at the test facility from 2010 to 2022, 684 control group monkeys were investigated. Among them, 3 animals in the 4-week toxicity studies and 5 animals in the 13-week toxicity studies showed a weight change rate of less than -10%, resulting in an incidence rate of 1.2%. However, these animals had adequate food consumption. Animals in the 4-week toxicity studies showed signs of stress in histopathology. Additionally, 2/3 animals in the 4-week toxicity studies had decreased blood glucose levels and 1/5 animal in the 13-week toxicity study fell into a crouching posture, suggesting hypoglycemia that was alleviated with glucose administration, indicating stress-induced metabolic abnormalities. From 2015, an enrichment program was implemented to improve animal welfare. Prior to this program, 2.4% of animals showed a weight change rate of less than -10%, which dropped to 0.25% post-implementation, suggesting the program's effectiveness in reducing stress. These results clarify the characteristics of animals that lose weight during toxicity studies and suggest that improving animal welfare can reduce the incidence rate.

Repeated surgical oocyte collection in mice enables the harvest of multiple cohorts of eggs from a single female, thereby reducing the need for animal use. Traditionally, oocyte collection for the mouse requires euthanasia, and only two sequential collections have been reported. The most efficient superovulation protocol involves the use of inhibin antiserum; however, repeated administration may pose a risk of anaphylactic reactions. In this study, we used severely immunodeficient NOG mice to evaluate the feasibility of performing three serial oocyte collections with inhibin antiserum treatments. We found that three consecutive cycles of antiserum administration followed by surgical retrieval cycles were achieved without mortality. Although the number of mice from which oocytes could be collected decreased over successive cycles, oocyte retrieval remained feasible. The application of the anti-adhesion film improved the number of mice from which oocytes could be recovered. Furthermore, embryos derived from the third retrieval developed to term after transfer. However, compared with the control group, we were unable to achieve a proportional increase in the number of retrieved oocytes corresponding to the number of collection cycles. Further improvements to this repeated collection method may include increasing the number of consecutive retrievals, optimizing the protocol to enhance both the oocytes yield and the fertilization rates. Such advances could increase oocyte yield per female, enable the mass production of embryos from fewer animals.

Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant neurological disorder caused by mutations in KCNC3. Our previous studies revealed that KCNC3 (Potassium Voltage-Gated Channel Subfamily C Member 3) mutation R423H results in an early-onset form of SCA13. Previous biological models of SCA13 include zebrafish and Drosophila but no mammalian systems. More recently, mouse models with Kcnc3 mutations presented behavioral abnormalities but without obvious pathological changes in the cerebellum, a hallmark of patients with SCA13. Here, we present a novel transgenic mouse model by bacterial artificial chromosome (BAC) recombineering to express the full-length mouse Kcnc3 expressing the R424H mutation. This BAC-R424H mice exhibited behavioral and pathological changes mimicking the clinical phenotype of the disease. The BAC-R424H mice (homologous to R423H in human) developed early onset clinical symptoms with aberrant gait, tremor, and cerebellar atrophy. Histopathological analysis of the cerebellum in BAC-R424H mice showed progressive Purkinje cell loss and thinning of the molecular cell layer. Additionally, Purkinje cells of BAC-R424H mice showed significantly lower spontaneous firing frequency with a corresponding increase in inter-spike interval compared to that of wild-type mice. Our SCA13 transgenic mice recapitulate both neuropathological and behavioral changes manifested in human SCA13 R423H patients and provide an advantageous approach to understanding the role of voltage-gated potassium channel in cerebellar morphogenesis and function. This mammalian in vivo model will lead to further understanding of the R423H allelic form of SCA13 from the molecular to the behavioral level and serve as a platform for testing potential therapeutic compounds.

DNA double-strand breaks (DSBs) are among the most hazardous cellular damages,potentially leading to cell death or oncogenesis if unrepaired. Genome editing methods,such as the CRISPR/Cas9 system, induce DSBs and utilize these repair pathways for geneknockout and knock-in. Although ionizing radiation also induces DSBs, it is not clearwhether the efficiency of genome editing is affected by ionizing radiation. This studyinvestigated the impact of gamma-ray exposure on the genome editing efficiency of theimproved genome editing via oviductal nucleic acid delivery (i-GONAD)method. Gamma-rays were exposed to pregnant mice receiving i-GONADtargeting the Hr gene, whose mutation causes hair loss in mice. Theexposure on the fertilization day (Day 0) decreased natural delivery rates and littersizes, with notable effects at 0.3 Gy or higher. Although the proportions of hairlessoffspring obtained by i-GONAD differed greatly between single-guide RNAs(sgRNAs) used, total mutation rates, including hairless, mosaic, and indel, wereequivalent. Gamma-ray exposure on Day 0 and the day after fertilization (Day 1) similarlyand almost dose-dependently enhanced the genome editing efficiency evaluated by the totalmutation rate. This study suggests the improvement of genome editing efficiency bygamma-ray exposure, at least in i-GONAD method, potentially facilitatingthe creation of diverse experimental animal models.

Heart failure (HF) is a clinical syndrome related to multiple causes, including oxidative stress. Acyl-CoA thioesterase 1 (ACOT1) is an enzyme in fatty acids metabolism, but it remains unclear in HF. Transverse aortic coarctation induced HF mouse model and hypoxia-stimulated cardiomyocyte (HL-1) model were established. ACOT1 expression was down-regulated in heart tissues of HF mice. Adeno-associated virus serotype 9 (AAV9)-mediated ACOT1 overexpression improved cardiac function and pathological injury of heart tissues in transverse aortic coarctation (TAC)-induced HF mice. ACOT1 overexpression ameliorated oxidative stress in heart tissues of HF mice and hypoxia-stimulated HL-1 cells, as indicated by reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels and elevated superoxide dismutase (SOD) and glutathione (GSH) levels. We found that ACOT1 overexpression inhibited apoptosis both in vivo and in vitro, with decreased protein levels of cleaved PARP, cleaved CASPASE-3, and cleaved CASPASE-9. Mechanically, ACOT1 activated Kelch-like ECH-associated protein1-NF-E2-related factor2 (KEAP1-NRF2) pathway, leading to the nuclear translocation of NRF2 and increased NRF2-regulated gene Nqo1 expression. Rescue experiment indicated that ML385 (NRF2 inhibitor) abolished the effect of ACOT1 overexpression on oxidative stress. Collectively, these results suggested that ACOT1 overexpression protects heart from injury by inhibiting oxidative stress and apoptosis, possibly through activating KEAP1-NRF2 pathway.

Physiological responses to inhaled anesthetics vary among species. Therefore, a precise anesthetic technique is important for each individual species. In this study, we focused on the degu (Octodon degus), a small herbivorous rodent. Degus have recently begun to be used as laboratory models for brain research because of certain human-like characteristics, such as spontaneous development of Alzheimer's disease. In this study, we evaluated appropriate induction and maintenance anesthesia conditions for isoflurane and sevoflurane in degus by a stimulation test, electroencephalography (EEG), minimum alveolar concentration (MAC), and vital signs. During induction, more rapid time to loss of the righting reflex and deeper anesthesia in degus were observed in isoflurane. The MAC value for degus were 1.75 ± 0.0% in isoflurane and 2.25 ± 0.27% in sevoflurane. Whereas some degus were awake during maintenance anesthesia using both anesthetics at concentrations of ≤2%, no rats were awake when using sevoflurane at a concentration of 2%. The duration of the total flat EEG, a measure of the depth of maintenance anesthesia, was longer for isoflurane than for sevoflurane. Furthermore, higher concentrations of both anesthetics suppressed the respiratory rate in degus. These new findings regarding inhalation anesthesia in degus will contribute to future developments in the fields of laboratory animals and veterinary medicine.

The Cynomolgus monkey is the most widely used models in non-clinical studies. As factors like age, gender, and breeding province may affect hematologic and serum biochemical parameters, it is important to establish base values of these parameters by these three factors and to determine the effects of these factors on the parameters. In total, 1794 Cynomolgus monkeys (Male: 901, Female: 893) were selected. A total of 24 hematologic and 21 serum biochemical parameters were measured, and the effects of age, gender, and breeding province were analyzed. Base values for hematologic and serum biochemical parameters were established by age, gender, and breeding province. A significant neutrophil percent, alkaline phosphatase, and creatinine differences were observed between different ages; a significant alkaline phosphatase, gamma glutamyl transpeptidase, and creatinine differences were observed between males and females; a significant lymphocyte percent, neutrophil percent, reticulocyte count, alkaline phosphatase, gamma glutamyl transpeptidase, and creatinine differences were observed between different breeding provinces. The results emphasize the importance of improving base values by age, gender, and breeding provinces. There was no statistically significant difference in most of the above parameters, and Cynomolgus monkeys from different breeding provinces can be used in the same study.

An unconventional myosin, myosin VI gene (MYO6), contributes to recessive and dominant hearing loss in humans and mice. The Kumamoto shaker/waltzer (ksv)mouse is a model of deafness resulting from a splice-site mutation in Myo6. While ksv/ksv homozygous mice are deaf due to cochlear haircell stereocilia fusion at the neonatal stage, the hearing phenotypes of ksv/+ heterozygous mice have been less clear. Due to this splicing error, most MYO6 proteinexpression is lost in ksv mice; however, MYO6 with a single amino acid mutation (p.E461K) remains expressed. In this study, we investigated the hearing phenotypes and effectof a p.E461K mutation in ksv/+ heterozygous mice. Hearing tests indicated that hearing loss in ksv/+ mice arises concurrently at both low and highfrequencies. In the low-frequency region, stereocilia fusions were detected in the inner hair cells of ksv/+ mice. Expression analysis revealed abnormal MYO6 expression andlocalization, along with atypical expression of proteins in the basal region of the stereocilia, suggesting that these abnormalities may contribute to stereocilia fusion inksv/+ mice. Conversely, although the expression patterns of MYO6 and stereociliary basal-region proteins appeared normal in the cochlear area corresponding tohigh-frequency sounds, stereocilia loss in the outer hair cells was observed in ksv/+ mice. These findings suggest that the ksv/+ mice exhibit distinctmechanisms underlying hearing loss across areas responsible for low- and high-frequency hearing, differing from those previously reported in heterozygous Myo6 mice withloss-of-function and missense mutant alleles.