In this paper we describe the existence and consequences of subspecific and individual variation in the genetic make-up of house mice. The purpose is to illustrate forms of variation that are often neglected in discussions about animal care and experimental design. Towards this end, different inbred mouse strains as well as genetically selected mouse lines are compared in relation to their ecological origin. Firstly, the behaviour of BALB/c, C57BL/6J and CBA mice is described in relation to different habitats. Furthermore, their aggression is compared, as measured by two paradigms. It appears that some inbred lines (eg BALB/c and C57BL/6J) clearly show behaviour that reflects the functional adaptation to the natural habitats in which their ancestors lived. Other strains (eg CBA) show a lack of such behavioural adaptation and their phenotypes appear to be very unstable over time. Secondly, two fundamentally different characters, both present in populations of wild house mice and under genetic control, are described: on the one hand, active copers are characterized by aggressive behaviour; on the other hand, passive copers are reluctant to attack. The active, aggressive animals (manipulators) are well adapted to an invariant environment like their own territory, whereas the passive, non-aggressive copers (adjustors) are well adapted to a changing environment, eg when roaming. We discuss to what extent these coping styles are present in laboratory strains of mice. The major conclusion with regard to both phenomena is that individual and subspecific variation may have significant implications for experimental design and the welfare of the experimental animals.

The present experiment compared the fear-related behaviour of a mouse strain selected over 101 generations for high litter size with that of a randomly selected strain. The H-strain, selected for large litter size, has a mean (± SD) litter size at birth of 21.5 ± 3.5 pups. The randomly bred C-strain has a mean (± SD) litter size of 9.6 ± 2.2 pups. The elevated plus-maze, the light:dark test and a resident:intruder test were used to measure how the mice responded to novelty. In the elevated plus-maze, a well-validated model of animal anxiety, the H-strain was significantly more anxious (having a lower percentage of entries into open arms) than the C-strain at 9 weeks of age. In the light: dark test, in which the light levels were similar to those in the home environment, the H-strain did not differ significantly from the C-strain in its avoidance of the brightest area. In the resident: intruder test, where aggression-trained, older H-strain males were the residents, 11-week-old intruding mice of the C-strain spent a higher percentage of their time in flight and immobility than intruders of the H-strain. There were clear anxiety- and fear-related differences between the strains, which may be related to their selection history. The results illustrate a need for further studies on the consequences of selection for increased production for the ability of animals to adapt to their home environment and cope with environmental changes.

Housing conditions can alter both the physiology and behaviour of laboratory animals. Forced-air-ventilated micro-isolation systems increase the efficient use of space, decrease the incidence of disease among laboratory rodents, and provide better working conditions for animal care staff; however, such systems can increase breeding variability and mortality. We examined the possibility that stressors associated with automated housing conditions evoke subtle changes among immune, endocrine, and behavioural parameters in mice housed in a static versus a forced-air-ventilated micro-isolation system. In addition, we assessed the effects of housing in the forced-air-ventilated micro-isolation system both with and without the use of an automatic watering system. Housing in the forced-air-ventilated micro-isolation system, using the automatic watering system, suppressed delayed-type hypersensitivity (DTH) responses, a measure of cell mediated immune function, compared with the responses of mice housed in static cages. Hypothalamic–pituitary–adrenal axis function was also altered by housing in the forced-air-ventilated micro-isolation system with the use of the automatic watering system, such that mice in this housing system had lower resting corticosterone concentrations and increased reactivity to restraint. Despite these changes in corticosterone, housing condition did not alter activity level or exploratory, anxiety-like, or depressive-like behaviours. These results suggest that investigators should carefully consider housing conditions in studies of immune and endocrine function.

Environmental light-dark cycles play an important role in behavioural and physiological processes. It is essential that laboratory vivaria be designed to properly control the light conditions in which laboratory mice are housed; however, this is not universally the case. Some laboratory vivarium doors are designed with windows, which allow light from the hallways to leak into the housing space during the rodents’ dark phase. Personnel entering and exiting the housing space during the dark phase can also create excessive light leak from brightly illuminated hallways. In this study, we investigated the hypothesis that exposure to dim light at night, as commonly experienced in many laboratory rodent housing spaces, alters mouse (Mus musculus) behaviour. We specifically analysed patterns of locomotor activity, anxiety- and depressive-like responses. Exposure to dim (5 lux) light at night altered home-cage locomotor activity and increased anxiety and some depressive responses among laboratory mice. These results suggest that light conditions can alter mouse behaviour and potentially influence experimental outcomes. Increased care should be taken to properly control light-dark conditions for laboratory animals.

A high circulating cholesterol concentration is considered an important risk factor for the development of cardiovascular disease. Since lean fish intake and fish protein supplementation have been associated with lower cholesterol concentration in some but not all clinical studies, the main aim of this study was to investigate the effect of diets containing proteins from fish muscles and fish byproducts on the serum/plasma TC concentration in rodents. A systematic literature search was performed using the databases PubMed, Web of Science and Embase, structured around the population (rodents), intervention (type of fish and fraction, protein dose, duration), comparator (casein as control protein) and the primary outcome (circulating total cholesterol). Articles were assessed for risk of bias using the SYRCLE’s tool. A meta-analysis was conducted in Review Manager v. 5.4.1 (the Cochrane Collaboration) to determine the effectiveness of proteins from fish on the circulating TC concentration. Thirty-nine articles were included in the systematic review and meta-analysis, with data from 935 rodents. The risk of bias is unclear since few of the entries in the SYRCLE’s tool were addressed. Consumption of proteins from fish resulted in a significantly lower circulating TC concentration when compared to control groups (mean difference -0.24 mmol/l, 95% confidence interval -0.34, -0.15, P < 0.00001), with high statistical heterogeneity (I2 = 71%). To conclude, proteins from fish muscles and byproducts show promise as a functional dietary ingredient or supplement by preventing high cholesterol concentration in rodents, thus reducing one of the most important risk factors for developing cardiovascular disease.

We show that in many extender models, e.g., the minimal one with infinitely many Woodin cardinals or the minimal with a Woodin cardinal that is a limit of Woodin cardinals, there are no generic embeddings with critical point $\omega _1$ that resemble the stationary tower at the second Woodin cardinal. The meaning of “resemble” is made precise in the paper (see Definition 0.3).

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

Polycystic ovary syndrome (PCOS) is a complex disorder and genetic factors are believed to play a role. The main aim was to investigate expression levels of genes involved in PI3K/AKT signalling pathway pretreatment and post-treatment. Mouse models of PCOS were generated. Group one included control mice with no polycystic ovaries (n = 4), Group 2 included a PCOS mouse model (n = 8), Group 3 included PCOS mice treated with clomiphene citrate (n = 7) and Group 4 included PCOS mice treated with clomiphene citrate, metformin and pioglitazone (n = 8). Histochemical analyses were performed. Total RNA was extracted and cDNA was synthesized. Irs, Akt1 and Akt2, mTor and Pdpk1 gene expression levels were evaluated by RT-PCR amplification. In Group 1, cortex and medulla were evaluated as normal; in Group 2, ovarian cortex was composed of immature oocytes and cystic follicles with atretic follicles. In Groups 3 and 4, follicles were in the process of normal follicle differentiation. The expression levels of Akt1 and Pi3k were significantly different (P < 0.0001) between Groups 1 and 2. The significant differences in expression levels of Pi3k and Akt1 were also observed between the Group 1 and both Groups 3 and 4 (P < 0.0001). Furthermore, significant variations of the expression levels of mTor between Groups 1 and 4 were observed. The extrapolation of results of this study may imply that follicular development may be regulated by molecular pathways involving Pi3k, Akt1 and mTor expression. Therefore, genes in the PI3K/AKT pathway may have a direct regulatory role in the development of PCOS.

Methomyl is a broad-spectrum carbamate insecticide that has a variety of toxic effects on humans and animals. However, there have been no studies on the toxicity of methomyl in female mammalian oocytes. This study investigated the toxic effects of environmental oestrogen methomyl exposure on mouse oocyte maturation and its possible mechanisms. Our results indicated that methomyl exposure inhibited polar body extrusion in mouse oocytes. Compared with that in the control group, in the methomyl treatment group, superoxide anion free radicals in oocytes were significantly increased. In addition, the mitochondrial membrane potential of metaphase II stage oocytes in the methomyl treatment group was significantly decreased, resulting in reduced mouse oocyte quality. After 8.5 h of exposure to methomyl, metaphase I stage mouse oocytes displayed an abnormal spindle morphology. mRNA expression of the pro-apoptotic genes Bax and Caspase-3 in methomyl-treated oocytes increased, which confirmed the apoptosis. Collectively, our results indicated that mouse oocyte maturation is defective after methomyl treatment at least through disruption of spindle morphology, mitochondrial function and by induction of oxidative stress.

The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

While metabolic disorders such as obesity and diabetes are costly and deadly to the current population, they are also extremely detrimental to the next generation. Much of the current literature focuses on the negative impact of poor maternal choices on offspring disease, while there is little work examining maternal behaviors that may improve offspring health. Research has shown that voluntary maternal exercise in mouse models improves metabolic function in offspring. In this study, we hypothesized that controlled maternal exercise in a mouse model will effect positive change on offspring obesity and glucose homeostasis. Female mice were separated into three groups: home cage, sedentary, and exercise. The sedentary home cage group was not removed from the home cage, while the sedentary wheel group was removed from the cage and placed in an immobile wheel apparatus. The exercise group was removed from the home cage and run on the same wheel apparatus but with the motor activated at 5–10 m/min for 1 h/d prior to and during pregnancy. Offspring were subjected to oral glucose tolerance testing and body composition analysis. There was no significant difference in offspring glucose tolerance or body composition as a consequence of the maternal exercise intervention compared to the sedentary wheel group. There were no marked negative consequences of the maternal controlled exercise intervention. Further research should clarify the potential advantages of the controlled exercise model and improve experimental techniques to facilitate translation of this research to human applications.

We analyze the hereditarily ordinal definable sets $\operatorname {HOD} $ in $M_n(x)[g]$ for a Turing cone of reals x, where $M_n(x)$ is the canonical inner model with n Woodin cardinals build over x and g is generic over $M_n(x)$ for the Lévy collapse up to its bottom inaccessible cardinal. We prove that assuming $\boldsymbol \Pi ^1_{n+2}$ -determinacy, for a Turing cone of reals x, $\operatorname {HOD} ^{M_n(x)[g]} = M_n(\mathcal {M}_{\infty } | \kappa _{\infty }, \Lambda ),$ where $\mathcal {M}_{\infty }$ is a direct limit of iterates of $M_{n+1}$ , $\delta _{\infty }$ is the least Woodin cardinal in $\mathcal {M}_{\infty }$ , $\kappa _{\infty }$ is the least inaccessible cardinal in $\mathcal {M}_{\infty }$ above $\delta _{\infty }$ , and $\Lambda $ is a partial iteration strategy for $\mathcal {M}_{\infty }$ . It will also be shown that under the same hypothesis $\operatorname {HOD}^{M_n(x)[g]} $ satisfies $\operatorname {GCH} $ .

A set of reals is universally Baire if all of its continuous preimages in topological spaces have the Baire property. ${\sf Sealing}$ is a type of generic absoluteness condition introduced by Woodin that asserts in strong terms that the theory of the universally Baire sets cannot be changed by set forcings. The ${\sf Largest\ Suslin\ Axiom}$ ( ${\sf LSA}$ ) is a determinacy axiom isolated by Woodin. It asserts that the largest Suslin cardinal is inaccessible for ordinal definable surjections. Let ${\sf LSA}$ - ${\sf over}$ - ${\sf uB}$ be the statement that in all (set) generic extensions there is a model of $\sf {LSA}$ whose Suslin, co-Suslin sets are the universally Baire sets. We outline the proof that over some mild large cardinal theory, $\sf {Sealing}$ is equiconsistent with $\sf {LSA}$ - $\sf {over}$ - $\sf {uB}$ . In fact, we isolate an exact theory (in the hierarchy of strategy mice) that is equiconsistent with both (see Definition 3.1). As a consequence, we obtain that $\sf {Sealing}$ is weaker than the theory “ $\sf {ZFC}$ + there is a Woodin cardinal which is a limit of Woodin cardinals.” This significantly improves upon the earlier consistency proof of $\sf {Sealing}$ by Woodin. A variation of $\sf {Sealing}$ , called $\sf {Tower \ Sealing}$ , is also shown to be equiconsistent with $\sf {Sealing}$ over the same large cardinal theory. We also outline the proof that if V has a proper class of Woodin cardinals, a strong cardinal, and a generically universally Baire iteration strategy, then $\sf {Sealing}$ holds after collapsing the successor of the least strong cardinal to be countable. This result is complementary to the aforementioned equiconsistency result, where it is shown that $\sf {Sealing}$ holds in a generic extension of a certain minimal universe. This theorem is more general in that no minimal assumption is needed. A corollary of this is that $\sf {LSA}$ - $\sf {over}$ - $\sf {uB}$ is not equivalent to $\sf {Sealing}$ .

Methomyl is a widely used carbamate insecticide and environmental oestrogen that has adverse effects on the reproductive system. However, there have been no reports on the effect of methomyl on early embryos in mammals. In this study, we explored the effect of methomyl exposure on the quality of early embryonic development in mice and the possible mechanisms. During in vitro culture, different concentrations of methomyl (10, 20, 30 and 35 μM) were added to mouse zygote medium. The results showed that methomyl had an adverse effect on early embryonic development. Compared with the control group, the addition of 30 μM methomyl significantly reduced the rate of early embryo blastocyst formation. Methomyl exposure can increase oxidative stress and impair mitochondrial function, which may be the cause of blastocyst formation. In addition, we found that methomyl exposure promoted apoptosis and autophagy in mouse blastocysts. The toxic effect of methomyl on early embryos may be the result of oxidative stress induction. Taken together, our results indicate that methomyl can cause embryonic development defects in mice, thereby reducing the quality of early embryo development.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

The aims of this research communication were to investigate the in vivo tissue accumulation of phytanic acid (PA) and any changes in the tissue fatty acid profiles in mice. Previous in vitro studies have demonstrated that PA is a milk component with the potential to cause both beneficial effects on lipid and glucose metabolism and detrimental effects on neuronal cells. However, there is limited information about its in vivo actions. In this study, mice were fed diets containing either 0.00 or 0.05% 3RS, 7R, 11R-PA, which is the isomer found in milk and the human body. After 4 weeks, adipose tissue, liver and brain were harvested and their fatty acid profiles were determined by gas chromatographic analysis. The results showed that PA and its metabolite pristanic acid accumulated in the adipose tissue of PA-fed mice, and that dietary PA decreased the hepatic compositions of several saturated fatty acids such as palmitic acid while increasing the compositions of polyunsaturated fatty acids including linoleic acid and docosahexaenoic acid. However, dietary PA neither accumulated nor had a high impact on the fatty acid profile in the brain. These results suggested that dietary PA could exert its biological activities in adipose tissue and liver, although the brain is relatively less affected by dietary PA. These data provide a basis for understanding the in vivo physiological actions of PA.

The aim of the study was to investigate the continuous changing pattern of H4K12 acetylation, and the expression levels of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) in mouse oocytes during meiosis and after parthenogenetic activation (PA). The immunofluorescence results showed hyperacetylation of lysine-12 on histone H4 (H4K12) in the germinal vesicle (GV) oocytes that then decreased during germinal vesicle breakdown (GVBD), and disappeared in metaphase II (MII). However, it reappeared in the early 1-cell embryos derived after 4 h of PA. The expression levels of some selected HATs and HDACs also validated the changing pattern of H4K12 acetylation during meiosis and PA. In conclusion, H4K12 is deacetylated in GVBD and MII, and re-hyperacetylated after PA.

In vitro activation of primordial follicles provides cancer patients subjected to oncotherapy with a safe therapeutic strategy for fertility preservation, however a successful protocol for activation of primordial follicles in prepubertal patients has not yet been defined comprehensively. There is evidence that amino acids such as leucine, arginine and glutamine could stimulate the mammalian target of rapamycin (mTOR) pathway, which plays a pivotal role in primordial follicle activation. Nevertheless, there has been no report that elucidates the effect of these amino acids on in vitro development of ovarian follicles. Therefore, the present study was conducted to evaluate the effects of these amino acids and their combination on the formation and activation of primordial follicles in 1-day-old murine ovaries during an 11-day culture period. The experimental groups consisted of base medium (BM), base medium + arginine (ARG), base medium + glutamine (GLU), base medium + leucine (LEU) and base medium + a combination of arginine, glutamine and leucine (AGL). The proportions of different stages of ovarian follicles and gene expression of regulatory factors were assessed using histology and quantitative real-time PCR on days 5 and 11 of culture. The proportion of transitional and primary follicles was greater in all amino acid-treated groups compared with the BM group (P < 0.05). Moreover, leucine resulted in elevated expression of Gdf9 and Bmp15, and glutamine augmented the expression of Pi3k on day 11 of culture. In conclusion, the present study showed that inclusion of leucine, glutamine, arginine or their combination in the culture medium for murine ovarian tissue could accelerate the activation of primordial follicles and alter the expression of the corresponding factors.

Polyploids generated by natural whole genome duplication have served as a dynamic force in vertebrate evolution. As evidence for evolution, polyploid organisms exist generally, however there have been no reports of polyploid organisms in mammals. In mice, polyploid embryos under normal culture conditions normally develop to the blastocyst stage. Nevertheless, most tetraploid embryos degenerate after implantation, indicating that whole genome duplication produces harmful effects on normal development in mice. Most previous research on polyploidy has mainly focused on tetraploid embryos. Analysis of various ploidy outcomes is important to comprehend the effects of polyploidization on embryo development. The purpose of this present study was to discover the extent of the polyploidization effect on implantation and development in post-implantation embryos. This paper describes for the first time an octaploid embryo implanted in mice despite hyper-polyploidization, and indicates that these mammalian embryos have the ability to implant, and even develop, despite the harmfulness of extreme whole genome duplication.