Bacteriophage-driven emergence and expansion of Staphylococcus aureus in rodent populations.

Human activities such as agriculturalization and domestication have led to the emergence of many new pathogens via host-switching events between humans, domesticated and wild animals. Staphylococcus aureus is a multi-host opportunistic pathogen with a global healthcare and economic burden. Recently, it was discovered that laboratory and wild rodents can be colonised and infected with S. aureus, but the origins and zoonotic potential of rodent S. aureus is unknown. In order to trace their evolutionary history, we employed a dataset of 1249 S. aureus genome sequences including 393 of isolates from rodents and other small mammals (including newly determined sequences for 305 isolates from 7 countries). Among laboratory mouse populations, we identified multiple widespread rodent-specific S. aureus clones that likely originated in humans. Phylogeographic analysis of the most common murine lineage CC88 suggests that it emerged in the 1980s in laboratory mouse facilities most likely in North America, from where it spread to institutions around the world, via the distribution of mice for research. In contrast, wild rodents (mice, voles, squirrels) were colonized with a unique complement of S. aureus lineages that are widely disseminated across Europe. In order to investigate the molecular basis for S. aureus adaptation to rodent hosts, genome-wide association analysis was carried out revealing a unique complement of bacteriophages associated with a rodent host ecology. Of note, we identified novel prophages and pathogenicity islands in rodent-derived S. aureus that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes promoting survival in new host-species.

Improving natural product research translation: From source to clinical trial.

While great interest in health effects of natural product (NP) including dietary supplements and foods persists, promising preclinical NP research is not consistently translating into actionable clinical trial (CT) outcomes. Generally considered the gold standard for assessing safety and efficacy, CTs, especially phase III CTs, are costly and require rigorous planning to optimize the value of the information obtained. More effective bridging from NP research to CT was the goal of a September, 2018 transdisciplinary workshop. Participants emphasized that replicability and likelihood of successful translation depend on rigor in experimental design, interpretation, and reporting across the continuum of NP research. Discussions spanned good practices for NP characterization and quality control; use and interpretation of models (computational through in vivo) with strong clinical predictive validity; controls for experimental artefacts, especially for in vitro interrogation of bioactivity and mechanisms of action; rigorous assessment and interpretation of prior research; transparency in all reporting; and prioritization of research questions. Natural product clinical trials prioritized based on rigorous, convergent supporting data and current public health needs are most likely to be informative and ultimately affect public health. Thoughtful, coordinated implementation of these practices should enhance the knowledge gained from future NP research.

Global spread of mouse-adapted Staphylococcus aureus lineages CC1, CC15, and CC88 among mouse breeding facilities.

We previously reported that laboratory mice from all global vendors are frequently colonized with Staphylococcus aureus (S. aureus). Genotyping of a snap sample of murine S. aureus isolates from Charles River, US, showed that mice were predominantly colonized with methicillin-sensitive CC88 strains. Here, we expanded our view and investigated whether laboratory mice from other global animal facilities are colonized with similar strains or novel S. aureus lineages, and whether the murine S. aureus isolates show features of host adaptation. In total, we genotyped 230 S. aureus isolates from various vendor facilities of laboratory mice around the globe (Charles River facilities in the USA, Canada, France, and Germany; another US facility) and university- or company-associated breeding facilities in Germany, China and New Zealand. Spa typing was performed to analyse the clonal relationship of the isolates. Moreover, multiplex PCRs were performed for human-specific virulence factors, the immune-evasion cluster (IEC) and superantigen genes (SAg). We found a total of 58 different spa types that clustered into 15 clonal complexes (CCs). Three of these S. aureus lineages had spread globally among laboratory mice and accounted for three quarters of the isolates: CC1 (13.5%), CC15 (14.3%), and CC88 (47.0%). Compared to human colonizing isolates of the same lineages, the murine isolates frequently lacked IEC genes and SAg genes on mobile genetic elements, implying long-term adaptation to the murine host. In conclusion, laboratory mice from various vendors are colonized with host-adapted S. aureus-strains of a few lineages, predominantly the CC88 lineage. S. aureus researchers must be cautioned that S. aureus colonization might be a relevant confounder in infection and vaccination studies and are therefore advised to screen their mice before experimentation.

The effects of shipping on early pregnancy in laboratory rats.

Although rats in various stages of pregnancy are routinely shipped by vendors, the effects of shipping on pregnancy outcomes have not been reported. This study examined the effects of shipping rats 1 day after mating. Two outbred stocks, (Crl:CD(SD), Crl:WI(Han)) and one inbred strain (F344/Crl) of rats (n=300/strain) were mated in a vendor barrier room at 3-month intervals five times, and either shipped the next day (total time in transit ∼24 hr) or held in the room of origin until parturition. The pregnancy status, length of gestation, number of pups born per female, sex ratio of pups born, and neonatal mortality were compared between transported and nontransported rats. These pregnancy and litter parameters were also compared among strains and examined for seasonality; no seasonal effects were observed. Neonatal mortality was negligible at less than 2% in any of the groups. All sex ratios were normal. Transportation affected pregnancy rates only in the F344/Crl, in which 81.8% of the nontransported versus 70% of the transported rats had pups (p=0.025). Overall, slightly fewer transported rats were pregnant, but they had larger litters (10.08 compared with 9.68, p=0.02, pooling across all three strains) so produced the same numbers of pups. A total of 77±8% of transported rats had gestation periods of 22 days or more compared with only 52±10% in the nontransported rats. The reason for larger litters in transported females is unclear. Longer gestation in transported females may be due to facultative embryonic diapause, which might have implications for reproductive toxicology.

Environmental Complexity and Research Outcomes.

Environmental complexity is an experimental paradigm as well as a potential part of animals' everyday housing experiences. In experimental uses, researchers add complexity to stimulate brain development, delay degenerative brain changes, elicit more naturalistic behaviors, and test learning and memory. Complexity can exacerbate or mitigate behavioral problems, give animals a sense of control, and allow for expression of highly driven, species-typical behaviors that can improve animal welfare. Complex environments should be designed thoughtfully with the animal's natural behaviors in mind, reported faithfully in the literature, and evaluated carefully for unexpected effects.

Tell-tale TINT: Does the Time to Incorporate into Nest Test Evaluate Postsurgical Pain or Welfare in Mice?

Identifying early indicators of distress in mice is difficult using either periodic monitoring or current technology. Likewise, poor pain identification remains a barrier to providing appropriate pain relief in many mouse models. The Time to Incorporate to Nest Test (TINT), a binary measure of the presence or absence of nesting behavior, was developed as a species-specific method of identifying moderate to severe distress and pain in mice. The current study was designed to evaluate alterations in nesting behavior after routine surgery and to validate the TINT's ability to measure pain-related behavioral changes. CD1 mice undergoing carotid artery catheterization as part of a commercial surgical cohort were randomly assigned various nesting, surgery, and analgesia conditions. To provide context for the TINT outcomes, we measured other variables affected by pain, such as weight loss, food consumption, and scores derived from the Mouse Grimace Scale (MGS). Mice that had surgery were more likely to have a negative TINT score as compared with controls. All mice were more likely to fail the TINT after receiving postoperative buprenorphine, suggesting that buprenorphine may have contributed to the failures. The TINT, MGS live scoring, and scoring MGS images all loaded strongly on a single component in a principal component analysis, indicating strong convergent validity between these measures. These data indicate that the TINT can provide a quick, objective indicator of altered welfare in mice, with the potential for a wide range of uses.

The epidemiology of fighting in group-housed laboratory mice.

Injurious home-cage aggression (fighting) in mice affects both animal welfare and scientific validity. It is arguably the most common potentially preventable morbidity in mouse facilities. Existing literature on mouse aggression almost exclusively examines territorial aggression induced by introducing a stimulus mouse into the home-cage of a singly housed mouse (i.e. the resident/intruder test). However, fighting occurring in mice living together in long-term groups under standard laboratory housing conditions has barely been studied. We performed a point-prevalence epidemiological survey of fighting at a research institution with an approximate 60,000 cage census. A subset of cages was sampled over the course of a year and factors potentially influencing home-cage fighting were recorded. Fighting was almost exclusively seen in group-housed male mice. Approximately 14% of group-housed male cages were observed with fighting animals in brief behavioral observations, but only 14% of those cages with fighting had skin injuries observable from cage-side. Thus simple cage-side checks may be missing the majority of fighting mice. Housing system (the combination of cage ventilation and bedding type), genetic background, time of year, cage location on the rack, and rack orientation in the room were significant risk factors predicting fighting. Of these predictors, only bedding type is easily manipulated to mitigate fighting. Cage ventilation and rack orientation often cannot be changed in modern vivaria, as they are baked in by cookie-cutter architectural approaches to facility design. This study emphasizes the need to invest in assessing the welfare costs of new housing and husbandry systems before implementing them.

Welfare Impact of Carbon Dioxide Euthanasia on Laboratory Mice and Rats: A Systematic Review.

Background: There has been increased concern about the suitability of CO2 as a method for euthanasia of laboratory mice and rats, including the potential discomfort, pain or distress that animals may experience prior to loss of consciousness; time to loss of consciousness; best methods for use of CO2; and the availability of better alternatives. These discussions have been useful in providing new information, but have resulted in significant confusion regarding the acceptability of CO2 for rodent euthanasia. In some cases, researchers and veterinarians have become uncertain as to which techniques to recommend or use for euthanasia of laboratory mice and rats. Methods: The International Association of Colleges of Laboratory Animal Medicine (IACLAM) convened a taskforce to examine the evidence for adverse welfare indicators in laboratory rats and mice undergoing CO2 euthanasia using a SYRCLE-registered systematic review protocol. Of 3,772 papers identified through a database search (PubMed, Web of Science, CAB Direct, Agricola, and grey literature) from 1900 to 2017, 37 studies were identified for detailed review (some including more than one species or age group), including 15 in adult mice, 21 in adult rats, and 5 in neonates of both species. Experiments or reports were excluded if they only assessed parameters other than those directly affecting animal welfare during CO2 induction and/or euthanasia. Results: Study design and outcome measures were highly variable and there was an unclear to high risk of bias in many of the published studies. Changes in the outcome measures evaluated were inconsistent or poorly differentiated. It is likely that repeated exposures to carbon dioxide inhalation are aversive to adult rats and mice, based on avoidance behavior studies; however, this effect is largely indistinguishable from aversion induced by repeated exposures to other inhalant anesthetic gasses. Conclusion: There is insufficient evidence to permit an unbiased assessment of the effect of CO2 inhalation during euthanasia on welfare indicators in laboratory mice and rats. Additional well-designed, unbiased, and adequately powered studies are needed to accurately assess the welfare of laboratory mice and rats undergoing euthanasia via CO2 gas.

Molecular Epidemiology of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in Wild, Captive and Laboratory Rats: Effect of Habitat on the Nasal S. aureus Population.

Rats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Free-living wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S.aureus lineages-many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S.aureus, respectively.

Ralstonia pickettii-induced ataxia in immunodeficient mice.

We report here the characterization of an asymmetric ataxia syndrome (head tilt and circling, with death in the most severe cases) demonstrated by profoundly immunodeficient mice housed at the Institut Curie SPF facility. The immune system of the affected mice had been genetically modified so that they were deficient in both B and T cells. Extensive bacteriologic, parasitic, serologic, and histopathologic analysis of the affected animals and their healthy controls led us to identify Ralstonia pickettii as the causative agent of the ataxic syndrome. The outbreak was managed through a test-and-cull process. Even though they also carried Ralstonia pickettii, immunocompetent mice that were kept in the same facility, did not show any of the signs that were expressed by their immunodeficient counterparts. This case highlights the difficulty of maintaining immunocompetent and immunodeficient mice in the same microbiologic unit and the importance of enlarging the spectrum of health monitoring to opportunistic agents when investigating clinical cases in populations of immunocompromised rodents.

Contemporary prevalence of infectious agents in laboratory mice and rats.

Periodic health screening of rodents used in research is necessary due to the consequences of unwanted infections. One determinant of the risk of infection for any given agent is its prevalence; other factors being equal, a prevalent agent is more likely than a rare one to be introduced to a research facility and result in infection. As an indicator of contemporary prevalence in laboratory populations of rats and mice, the rate of positive results in the samples received at a major commercial rodent diagnostic laboratory was compiled for this paper. Although samples from laboratory rodent vendors have been excluded, results are tabulated from samples from more than 500,000 mice and 80,000 rats submitted over several years from pharmaceutical, biotechnology, academic, and governmental institutions in North America and Europe, allowing meaningful determination of which agents are common in the research environment versus which agents are rare. In mice, commonly detected infectious agents include mouse norovirus, the parvoviruses, mouse hepatitis virus, rotavirus, Theiler's murine encephalomyelitis virus, Helicobacter spp., Pasteurella pneumotropica, and pinworms. In rats, commonly detected infectious agents include 'rat respiratory virus', the parvoviruses, rat theilovirus, Helicobacter spp., P. pneumotropica, and pinworms. A risk-based allocation of health-monitoring resources should concentrate frequency and/or sample size on these high-risk agents, and monitor less frequently for the remaining, lower-risk, infectious agents.

Refining timed pregnancies in two strains of genetically engineered mice.

In order to efficiently generate genetically engineered mouse (GEM) fetuses or neonates of a specified age range, researchers must develop strain-specific strategies, including reliable early pregnancy detection. The authors evaluated pregnancy indices (pregnancy rate, plug rate, pregnant plugged rate, first litter size and body weight) in two GEM breeding colonies: homozygous soluble epoxide hydrolase knockout (sEHKO) mice (n=164 females) and L7-tau-green fluorescent protein (GFP) transgenic mice (n=61 females). The goals of the study were to determine the most accurate early pregnancy indicator and to reliably and cost-effectively produce timed pregnant females that were between gestation days 16 and 18. The authors set up each timed mating by placing two naturally synchronized females with a male for 48 h. When males were present, personnel checked each female daily for a vaginal plug. They then weighed the females immediately, 1 week and 2 weeks after removing the males. In both sEHKO and GFP colonies, increases in body weight at 1 and 2 weeks after timed male exposure more reliably and consistently indicated pregnancy than did plug detection. Further evaluations and protocol refinements are planned based on litter size and litter number in these colonies.

Using Hysterectomy Rederivation to Produce Guinea Pigs (Cavia porcellus) Free of Guinea Pig Cytomegalovirus.

Due to similarities in placentation, guinea pigs can be used as models of human cytomegalovirus infection, but they mustbe free of guinea pig cytomegalovirus. Many commercial guinea pig colonies are enzootically infected with guinea pigcytomegalovirus, which can be transmitted vertically as well as horizontally through saliva, vaginal secretions, and milk.These characteristics make its eradication in a commercial setting challenging. Because embryo transfer technology in guineapigs is in its infancy, it is not generally a viable option for obtaining animals free of guinea pig cytomegalovirus. However,a combination of hysterectomy rederivation and testing by PCR assay and serology of both dams and offspring from anenzootically infected colony produced offspring free of guinea pig cytomegalovirus.

Sorting it out: bedding particle size and nesting material processing method affect nest complexity.

As part of routine husbandry, an increasing number of laboratory mice receive nesting material in addition to standard bedding material in their cages. Nesting material improves health outcomes and physiological performance in mice that receive it. Providing usable nesting material uniformly and efficiently to various strains of mice remains a challenge. The aim of this study was to determine how bedding particle size, method of nesting material delivery, and processing of the nesting material before delivery affected nest building in mice of strong (BALB/cAnNCrl) and weak (C3H/HeNCrl) gathering abilities. Our data suggest that processing nesting material through a grinder in conjunction with bedding material, although convenient for provision of bedding with nesting material 'built-in', negatively affects the integrity of the nesting material and subsequent nest-building outcomes. We also found that C3H mice, previously thought to be poor nest builders, built similarly scored nests to those of BALB/c mice when provided with unprocessed nesting material. This was true even when nesting material was mixed into the bedding substrate. We also observed that when nesting material was mixed into the bedding substrate, mice of both strains would sort their bedding by particle size more often than if it were not mixed in. Our findings support the utility of the practice of distributing nesting material mixed in with bedding substrate, but not that of processing the nesting material with the bedding in order to mix them.

Aggression in group-housed laboratory mice: why can't we solve the problem?

Group housing is highly important for social animals. However, it can also give rise to aggression, one of the most serious welfare concerns in laboratory mouse husbandry. Severe fighting can lead to pain, injury and even death. In addition, working with animals that are severely socially stressed, wounded or singly-housed as a result of aggression may compromise scientific validity. Some general recommendations on how to minimize aggression exist, but the problem persists. Thus far, studies attempting to find solutions have mainly focused on social dominance and territorial behavior, but many other aspects of routine housing and husbandry that might influence aggressive behavior have been overlooked. The present way of housing laboratory mice is highly unnatural: mice are prevented from performing many species-typical behaviors and are routinely subjected to painful and aversive stimuli. Giving animals control over their environment is an important aspect of improving animal welfare and has been well-studied in the field of animal welfare science. How control over the environment influences aggression in laboratory mice, however, has not been closely examined. In this article, we challenge current ways of thinking and propose alternative perspectives that we hope will lead to an enhanced understanding of aggression in laboratory mice.

Introducing Therioepistemology: the study of how knowledge is gained from animal research.

This focus issue of Lab Animal coincides with a tipping point in biomedical research. For the first time, the scale of the reproducibility and translatability crisis is widely understood beyond the small cadre of researchers who have been studying it and the pharmaceutical and biotech companies who have been living it. Here we argue that an emerging literature, including the papers in this focus issue, has begun to congeal around a set of recurring themes, which themselves represent a paradigm shift. This paradigm shift can be characterized at the micro level as a shift from asking "what have we controlled for in this model?" to asking "what have we chosen to ignore in this model, and at what cost?" At the macro level, it is a shift from viewing animals as tools (the furry test tube), to viewing them as patients in an equivalent human medical study. We feel that we are witnessing the birth of a new discipline, which we term Therioepistemology, or the study of how knowledge is gained from animal research. In this paper, we outline six questions that serve as a heuristic for critically evaluating animal-based biomedical research from a therioepistemological perspective. These six questions sketch out the broad reaches of this new discipline, though they may change or be added to as this field evolves. Ultimately, by formalizing therioepistemology as a discipline, we can begin to discuss best practices that will improve the reproducibility and translatability of animal-based research, with concomitant benefits in terms of human health and animal well-being.

Review of CO2 as a Euthanasia Agent for Laboratory Rats and Mice.

Selecting an appropriate, effective euthanasia agent is controversial. Several recent publications provide clarity on the use of CO2 in laboratory rats and mice. This review examines previous studies on CO2 euthanasia and presents the current body of knowledge on the subject. Potential areas for further investigation and recommendations are provided.

The effect of early life experience, environment, and genetic factors on spontaneous home-cage aggression-related wounding in male C57BL/6 mice.

Aggression is a major welfare issue in mice, particularly when mice unfamiliar to each other are first placed in cages, as happens on receipt from a vendor, and following cage cleaning. Injuries from aggression are the second leading cause of unplanned euthanasia in mice, following ulcerative dermatitis. Commonly employed strategies for reducing aggression-related injury are largely anecdotal, and may even be counterproductive. Here we report a series of experiments testing potential explanations and interventions for post-shipping aggression-related injuries in C57BL/6 mice. First, we examined the effects of weaning: testing whether manipulating weaning age reduced aggression-related injuries, and if repeated mixing of weaned mice before shipping increased these injuries. Contrary to our predictions, repeated mixing did not increase post-shipping injurious aggression, and early weaning reduced aggression-related injuries. Second, we examined potential post-shipping interventions: testing whether lavender essential oil applied to the cage reduced aggression-related injuries, and whether a variety of enrichments decreased injurious aggression. Again, contrary to predictions, lavender increased wounding, and none of the enrichments reduced it. However, consistent with the effects of weaning age in the first experiment, cages with higher mean body weight showed elevated levels of aggression-related wounding. Finally, we tested whether C57BL/6 substrains and identification methods affected levels of intra-cage wounding from aggression. We found no effect of strain, but cages where mice were ear-notched for identification showed higher levels of wounding than cages where mice were tail-tattooed. Overall, these results emphasize the multifactorial nature of home-cage injurious aggression, and the importance of testing received wisdom when it comes to managing complex behavioral and welfare problems. In terms of practical recommendations to reduce aggressive wounding in the home cage, tail tattooing is recommended over ear notching and late weaning should be avoided.

Laboratory Mice Are Frequently Colonized with Staphylococcus aureus and Mount a Systemic Immune Response-Note of Caution for In vivo Infection Experiments.

Whether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. We previously identified a mouse-adapted S. aureus strain, which caused infections in laboratory mice. This raised the question whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments. Publicly available health reports from commercial vendors revealed that S. aureus colonization is rather frequent, with rates as high as 21% among specific-pathogen-free mice. In animal facilities, S. aureus was readily transmitted from parents to offspring, which became persistently colonized. Among 99 murine S. aureus isolates from Charles River Laboratories half belonged to the lineage CC88 (54.5%), followed by CC15, CC5, CC188, and CC8. A comparison of human and murine S. aureus isolates revealed features of host adaptation. In detail, murine strains lacked hlb-converting phages and superantigen-encoding mobile genetic elements, and were frequently ampicillin-sensitive. Moreover, murine CC88 isolates coagulated mouse plasma faster than human CC88 isolates. Importantly, S. aureus colonization clearly primed the murine immune system, inducing a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates. Phospholipase C emerged as a promising test antigen for monitoring S. aureus colonization in laboratory mice. In conclusion, laboratory mice are natural hosts of S. aureus and therefore, could provide better infection models than previously assumed. Pre-exposure to the bacteria is a possible confounder in S. aureus infection and vaccination studies and should be monitored.

Two of a Kind or a Full House? Reproductive Suppression and Alloparenting in Laboratory Mice.

Alloparenting, a behavior in which individuals other than the actual parents act in a parental role, is seen in many mammals, including house mice. In wild house mice, alloparental care is only seen when familiar sibling females simultaneously immigrate to a male's territory, so in the laboratory, when a pair of unfamiliar female wild mice are mated with a male, alloparenting does not occur because one female will typically be reproductively suppressed. In contrast, laboratory mice are assumed to alloparent regardless of familiarity or relatedness and are therefore routinely trio bred to increase productivity. Empirical evidence supporting the presence of alloparental care in laboratory mice is lacking. Albino and pigmented inbred mice of the strain C57BL/6NCrl (B6) and outbred mice of the stock Crl:CF1 (CF1) were used to investigate alloparenting in laboratory mice since by mating pigmented and albino females with albino males of the same stock or strain, maternal parentage was easily determined. We housed pairs (M:F) or trios (M:2F) of mice in individually ventilated cages containing nesting material and followed reproductive performance for 16 weeks. Females in trios were tested to determine dominance at the start of the experiment, and again 5 days after the birth of a litter to determine if a female's dominance shifted with the birth of pups. Results showed a significant and expected difference in number of offspring produced by B6 and CF1 (p < 0.0001). Pigmented mice nursed and nested with albino pups and vice-versa, confirming empirical observations from many that group nesting and alloparenting occurs in unrelated laboratory mice. When overall production of both individual mice and cages was examined, reproductive suppression was seen in trio cages. Dominance testing with the tube test did not correlate female reproduction with female dominance in a female-female dyad. Due to the reproductive suppression noted in trios, on a per-mouse basis, pair mating outperformed trio mating (p = 0.02) when the measure was weaned pups/female/week. No infanticide was seen in any cages, so the mechanism of reproductive suppression in trio matings may occur before birth.