Influencing and Regulating the Estrous Cycle in Outbred CD1 Laboratory Mice (Mus musculus)
The Whitten effect is a widely used tool for manipulating the mouse estrous cycle and generating reproductively active females within the laboratory setting. Typically, peak numbers of sexually receptive mice occur following exposure to male pheromones, resulting in a higher number of successful copulations on the third day after exposure. Although this method has improved efficiencies, the percentage of females mated and subsequently deemed to be pregnant/pseudopregnant remains relatively low, around 50%. In experiment 1, we aimed to 1) further understand cyclicity; 2) determine whether the initial cycle stage plays an importance on day 3 receptivity; and 3) identify any repetitive patterns/cycle stabilization. Mice (n = 27) were assigned to group cages according to cycle stage (proestrus, estrus, metestrus, diestrus). Experiment 2 was developed to determine an optimum treatment to promote receptivity by exposure to various pheromone stimuli. Mice (n = 45) were randomly assigned to 5 treatment groups (PBS-treated sham soiled bedding, male soiled bedding, live male, pregnant females, and lactating females). In both experiments, daily vaginal cytology was performed for 21 days to determine the cycle stage. Results from experiment 1 indicate that the initial cycle stage did not contribute to day 3 receptivity, although synchronization within several groups/cages was noted, and that the greatest numbers of estrous animals were obtained on days 6 and 7. Experiment 2 revealed that exposure to live males and lactating females both significantly improved receptivity compared with the PBS, male soiled bedding, and pregnant female groups. These results indicate that current strategies used for routine synchronization could be further improved through alternative housing regimens without compromising animal welfare.Abstract
Introduction
The laboratory mouse (Mus musculus) is a polyestrous mammal that ovulates throughout the year with estrous cycles lasting around 4 to 5 days. Each cycle consists of 4 stages: proestrus, estrus, metestrus, and diestrus.2 Generally, mating is confined to the receptive stages of the cycle (proestrus and estrus),35 which, combined, last approximately 12 to 48 hours.37 Akin to other mammals, murine ovarian follicles develop in response to follicle-stimulating hormone release from the anterior pituitary, in turn induced by hypothalamus-derived gonadotropin-releasing hormone. While luteinizing hormone is released via the same pathway, its role is to prime ovarian follicles for ovulation.19 During proestrus, a surge of follicle-stimulating and luteinizing hormones induces rupture of mature follicles, expelling the oocytes into the oviduct for fertilization.8 At this time, female mice may be paired with fertile males with the aim to obtain staged embryos, fetuses, live pups, or they may be euthanized to collect unfertilized oocytes for in vitro fertilization. When females are mated with sterile males, they typically undergo a period of pseudopregnancy, a neuroendocrine state that replicates similar reproductive physiologic responses to those experienced in early pregnancy.44 This strategy enables the creation of surrogate embryo recipients for manipulated embryos and rederivation procedures in these animals in which changes in the hormonal milieu have primed the endometrium for embryo implantation.40
To create pseudopregnant embryo recipients for embryo transfer, females are typically housed with either genetically or surgically sterile males in a 1:1 mating ratio.16 To ensure that sufficient pseudopregnant recipients are available on the day of embryo transfer, a significantly larger number of these pairings are scheduled as not all matings result in successful copulation and subsequent pseudopregnancy. To compensate for this, 4 to 5 times as many females as those needed as recipients are set up with males,35 which results in very significant animal wastage.
Moreover, females used in these setups are not reused for 2 weeks following the initial pairing period (the duration of pseudopregnancy being 10 to 13 days),36 by which time they can have developed a body habitus less conducive to successful surgical embryo transfer. Although successful mating rates (confirmed by the presence of a copulation plug) can be improved by means of the Whitten effect, this still equates to around half of the animals being wasted.41 Strategies such as visual inspection of the vulva and vaginal cytology can improve the likelihood of a successful mating by selecting sexually receptive animals, thereby reducing the pool size of females set up. Nevertheless, an external influence is still required to synchronize large numbers of females.2
During the 1950s and the following 3 decades, a significant amount of research was committed to better understanding the mouse estrous cycle, its stages/states, and its governing influences.5,6,10,22,27,41 It was at the start of this period that the gold standard method of estrus induction was developed: the Whitten effect.41 Whitten effect-induced estrus in female mice is achieved using olfactory stimulation by exposing them to the priming pheromones found in male urine38 and results in the majority of copulations occurring on the third evening.43 It is now the standard method for estrous cycle synchronization worldwide. Other putative male urine chemosignal-driven effects were also discovered at this time, including the Bruce effect, where females carrying preimplantation embryos (following embryo transfer or fertile matings) discontinued their pregnancies through failed implantation following exposure to the scent of an unfamiliar male.6
Further work5,6,42 also highlighted the importance of olfactory female pheromone stimulation on females. For example, group housing of female mice for prolonged periods was shown to disrupt normal estrous cyclicity, resulting in periods of anestrus (the Lee-Boot effect).6,39,42 Although these phases can be overcome by the presence of male urinary chemosignals, female overcrowding can suppress any such male effect.6 Cyclicity can further be altered by olfactory signals from other females in the social environment that assist in the coordination of fertility and pup care.29 For example, urinary volatile ketones from pregnant/lactating mice have been shown to accelerate puberty in young females while promoting estrus in adults (the Hoover-Drickamer effect).12 Interestingly, these olfactory cues can nevertheless be negated by external parameters including population density, light cycle, nutrition, and social cues4 (e.g., ultrasonic vocalizations, which have been shown to be relevant during courtship,17,30 social support, and parental care31), which suggests that a well-balanced environment is required to produce regularly cycling female mice.
In recent years, the focus in laboratory mouse estrous cycle management has moved to the use of injectable gonadotropins, synthetic prostaglandins, and gonadotropin-releasing hormone stimulants as well as progesterone treatment.9,18,28,32 While regimens based on these methods have been shown to be effective in increasing the pool of induced estrous females,18 they nevertheless have significant drawbacks. For example, exogenous stimulant injections are typically time sensitive, require technical training, and place additional labor and management pressures on technical teams. Moreover, the use of many such injection-based regimens falls under the umbrella of regulated procedures [e.g., under the Animals (Scientific Procedures) Act, 1986]20 such that supernumerary mice cannot be reused thereafter for either breeding or experimental work, leading to significant wastage. In addition, the use of injectable gonadotropins, synthetic prostaglandins, and gonadotropin-releasing hormone stimulants has been shown to have detrimental effects on murine oocyte/embryo quality, implantation rates, and the reproductive status of generated female offspring.3,14,23,24 Given the increased emphasis placed on animal welfare (e.g., replacement, reduction, and refinement [the 3Rs]), there is a cogent argument to revisit the opportunity to develop physiologic strategies for the exogenous manipulation of the estrous cycle in mice. This study therefore aimed to investigate receptivity induction and synchronisation using non-invasive methods to produce large pools of estrous females.
Materials and Methods
All experiments were performed after local ethical approval from the Animal Welfare and Ethics Research Committee and project license approval under the Animals (Scientific Procedures) Act, 1986, with strict adherence to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes.
Experiment 1.
This experiment aimed to determine whether estrus could be induced by means of the Whitten effect to establish if animals adopted a predictable natural cycle pattern and, if so, when the peak number of estrous females would occur. Twenty-seven virgin female outbred CRL:CD1(ICR) mice aged 9 to 12 weeks weighing 28 to 43 g were obtained from the in-house colony at St. James’s Biomedical Services, University of Leeds, and housed in groups of 3 to 5 animals in GM500 Sealsafe Individually Ventilated Cages (Tecniplast, Buguggiate, Italy) with a floor area of 501 cm2 (Figure 1). CRL:CD1(ICR) mice were used since this is a proven robust outbred mouse strain with good fertility and pregnancy rates. This makes it an ideal recipient strain for embryo transfers,25 which has been used for over a decade, thereby generating an expansive archive of reproductive performance data. Mice were housed behind the specified pathogen-free barrier of the animal facility, which was routinely health screened following Federation of European Laboratory Animal Science Associations (FELASA) guidelines (Table S1). Cages remained in the same room as colony production to obviate any effects related to changes in the environment and working staff. Environmental conditions were maintained using an electronic building management system set to 21 to 23 °C, 50% relative humidity (±10%), and artificial lighting on a 12:12 h light/dark cycle (lights on at 0600; lights off at 1800). Low-volume radios were played during technician working hours. Animals had ad libitum access to filtered UV-treated water and a pelleted rodent diet [CRM (P) POLY IRR, Special Diet Services, Essex, UK]. Mice were housed on 3Rs bedding and given abundant sizzle nest nesting material and a mouse igloo (Datesand, Manchester, UK) for environmental enrichment. Cages were cleaned out fully every 8 days until the end of the study. All animals were handled using the cupping method and procedures were performed on a benchtop in a clean procedure room. Females had had no prior contact with males since weaning at age 19 to 21 days.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-044
Vaginal cytology was used to establish estrous cycle stage, a method shown to be more accurate than vulval visualization alone.7,8 On day 0, the vaginal opening was visually observed to determine the cycle stage before cytologic evaluation. Animals were placed on a corkboard covered with a clean tissue and gently restrained by placing the palm of the non-dominant hand across the dorsum and holding the base of the tail between the bottom of the thumb and forefinger to expose the vagina. Using a sterile Pasteur pipette (Starlab; E1414-0111), approximately 35 µL of Dulbecco PBS (Thermo Scientific; 14040-133) was flushed 3 to 5 times within the vaginal opening (2 to 3 mm deep), expelled onto a clean microscope slide (Scientific Laboratory Supplies; MIC3452), and observed immediately under an inverted microscope (Nikon; SMZ1500). The cycle stage was determined using the criteria set in 19221 and in 20127 (Figures 2 and 3). Mice were then group housed according to their day 0 cycle stage (as a standard baseline) in fresh clean cages: Proestrus (n = 10; divided into 2 cages of 5), Estrus (n = 3; in one cage), Metestrus (n = 5; in one cage), and Diestrus (n = 9; divided into one cage of 5 and one of 4). These same animals were also considered as a Pooled Group, regardless of estrous stage. Pheromone exposure was achieved by using soiled bedding from the CRL:CD1(ICR) vasectomized male colony. Twenty grams of male urine-soaked bedding was added to each cage on day 0, with further soiled bedding added on days 4, 8, 12, and 16 to mimic the normal 3-day mating protocol and following a typical 4 to 5 day estrous cycle. Vaginal cytology was repeated between 12 and 3 PM daily.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-044
Experiment 2.
The second experiment investigated the merits of alternative methods of estrus induction using various olfactory cues, with a particular focus on whether success rates obtained by exposure to male pheromones could be exceeded. Environmental conditions and feeding were as described for experiment 1. Forty-five virgin female outbred CRL:CD1(ICR) mice aged 6 weeks, weighing 23–30 g were obtained from Charles River UK, were randomly housed in groups of 9 across 5 GR1800 double-decker Sealsafe Individually Ventilated Cages and allowed to habituate for 2 weeks (Figure 4). Perforated metal cage dividers (Tecniplast UK Fabrications) were inserted with females housed on one side, giving a floor area of 931 cm2 plus the additional vertical surface offered by the cage divider for climbing activities. Experimental animals were housed on the right side of the divided cage, and the treatment was applied to the left. Due to the cages’ air circulation system, the clean air inlet was on the treatment side and exhausted on the experimental side, allowing maximum exposure to pheromonal stimuli. During this period, females had no exposure to any males or their pheromones. Based on the results of experiment 1, females were cytologically estrous cycle staged using the same vaginal cytology technique described above but randomly allocated to the different groups, which included the following: Sham Soiled Bedding, Male Soiled Bedding, Live Male, Pregnant Females, and Lactating Females. Sham soiled bedding involved using 20 g of clean bedding soaked with 20 mL PBS, while soiled bedding was as described for experiment 1 but obtained from the cages of CRL:CD1(ICR) sexually experienced, intact males; this was added on days 0, 4, 8, 12, and 16 for both groups. In the Live Male group, an experienced CRL:CD1(ICR) male aged 13 weeks from the in-house colony was added to the treatment side of the cage and maintained there throughout the duration of the study, enabling interaction but no direct contact through the cage divider. In the Pregnant Females group, 2 CRL:CD1(ICR) female mice aged 8 weeks were obtained from Charles River UK and placed with a male from the same strain aged 13 weeks from the in-house colony until a copulation plug was observed in each female (while housed in GM500 Sealsafe Individually Ventilated Cages within the animal facility’s quarantine suite). From 4 days postcoitum, these were added to the treatment side of the experimental cages. To maintain the pheromonal stimulus for the duration of the study, these were replaced with fresh pregnant mice generated in the same manner on day 16 of gestation. Finally, in the Lactating Females group, the 16 day gestation mice from the Pregnant Females group were placed into the treatment side of the cage and allowed to birth and nurse their pups for the duration of the study (i. e., for an additional 21 days) to minimize animal wastage. The 9 study females were added to the experimental side of the cage 4 days after their pregnant counterparts had given birth.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-044
For both experiments 1 and 2, female animals either in proestrus or estrus at the time of vaginal cytology were considered sexually receptive. All animal handling, husbandry, and experimental procedures were performed by a single operator (CF) to obviate the risk of interobserver variability in cytology interpretation in particular and to reduce any potential handler-related stressors.
Data presentation and statistical analyses.
Results are presented as means ± SE. Groups were compared using χ2 tests to establish receptivity levels over the 21 day period in both experiments 1 and 2. A P value of less than 0.05 was considered statistically significant. Longitudinal time-based assessments were assessed by Kruskall-Wallis tests. Statistical analyses were performed on IBM SPSS Statistics (Version 27).
Results
Experiment 1.
Significant differences in receptivity were observed on days 1 and 7 post-stimulus exposure (P < 0.05; Table 1). Cycle synchronization (defined as larger numbers of animals concurrently in the same stage of the estrous cycle) was most noticeable in the Estrus group (Figure 5). When all animals were pooled (Pooled Group that is independent of the estrous cycle stage at the start of the experiment), the largest number of receptive (that is proestrus/estrus) females was noted to occur on days 6 (74%) and 7 (81%), broadly mimicking the overall pattern noted in individual groups (Figure 6).
| Initial cycle stage (d) | No. receptive females (%) | ||||
|---|---|---|---|---|---|
| Proestrus | Estrus | Metestrus | Diestrus | χ2(df); P | |
| 0 | 10 (100) | 3 (100) | 0 (0) | 0 (0) | 27.003 |
| 1 | 8 (80) | 3 (100) | 1 (20) | 0 (0) | 17.283 |
| 2 | 7 (70) | 2 (67) | 4 (80) | 5 (56) | 0.953 |
| 3 | 4 (40) | 2 (67) | 3 (60) | 0 (0) | 7.803 |
| 4 | 6 (60) | 1 (33) | 1 (20) | 1 (11) | 5.603 |
| 5 | 6 (60) | 0 (0) | 0 (0) | 3 (33) | 7.203 |
| 6 | 6 (60) | 2 (67) | 3 (60) | 9 (100) | 4.783 |
| 7 | 5 (50) | 3 (100) | 5 (100) | 9 (100) | 10.433 |
| 8 | 5 (50) | 2 (67) | 4 (80) | 3 (33) | 3.103 |
| 9 | 3 (30) | 0 (0) | 2 (40) | 3 (33) | 1.583 |
| 10 | 5 (50) | 2 (67) | 2 (40) | 2 (22) | 2.473 |
| 11 | 6 (60) | 2 (67) | 3 (60) | 7 (78) | 0.803 |
| 12 | 5 (50) | 2 (67) | 4 (80) | 7 (78) | 2.153 |
| 13 | 2 (20) | 0 (0) | 2 (40) | 2 (22) | 1.803 |
| 14 | 4 (40) | 2 (67) | 1 (20) | 2 (22) | 2.603 |
| 15 | 6 (60) | 3 (100) | 2 (40) | 4 (44) | 3.423 |
| 16 | 8 (80) | 3 (100) | 2 (40) | 5 (56) | 4.403 |
| 17 | 7 (70) | 0 (0) | 2 (40) | 4 (44) | 4.883 |
| 18 | 2 (20) | 1 (33) | 2 (40) | 5 (56) | 2.613 |
| 19 | 3 (30) | 2 (67) | 3 (60) | 5 (56) | 2.213 |
| 20 | 5 (50) | 2 (67) | 2 (40) | 5 (56) | 0.613 |
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-044
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-044
Experiment 2.
Both the Live Male and Lactating Female groups showed a statistically significant increase in the overall number of days females were receptive over the study period (P < 0.05; Figure 7). Furthermore, a pairwise comparison indicated that there was no significant difference between the number of days receptive between the PBS Sham Soiled Bedding, Soiled Bedding, and Pregnant Female groups. No significant difference was observed between the Live Male and Lactating Females groups.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-044
Discussion
The purpose of this 2-part study was to consider non-invasive, physiologic approaches for the experimental manipulation of the estrous cycle in mice. Experiment 1 aimed to establish Whitten effect-based estrus induction and peak responses over multiple cycles over time. As highlighted by the Pooled Group, the estrous stage at which females received olfactory cues as part of the Whitten effect had a negligible effect on group synchrony. As anticipated, there was a peak in the number of receptive females on day 2, which reflects the timing of the original observations of Whitten et al.43 The number of receptive females at this point in time was 67%, which was in excess of the figures reported in the original study. By the end of the first week, all groups (except for the proestrus group) had adopted an evident, broadly synchronous 4- to 5-day cycle pattern2 (Figures 5 and 6) and produced a surge in the number of receptive females on day 7 (Table 1 and Figure S1). Importantly, the percentage of proestrus/estrus animals on this day (81%) markedly exceeded not only those achieved by Whitten41 but also those that were recorded on day 2.
Although animals subsequently maintained a cyclical pattern of receptivity peaks, levels as high as those noted on days 6 and 7 were not replicated thereafter. This observation may be due to synergistic factors, possibly the fact that these were young, virgin females with no previous exposure to male pheromones (wherein the animals would likely have been experiencing a mixture of normal cycling and anestrus) combined with the Lee-Boot effect under near ideal environmental conditions. Within this, using the Whitten effect 6 or 7 days in advance would allow the activation and regulation of cycles to increase the number of receptive females accordingly. Based on these findings (and if verifiable in a larger number of animals), we propose that to maximize the number of receptive animals induced by the Whitten effect, investigators should consider capturing females at the time of their second rather than their first estrus. This would only involve a small increase of 3 or 4 days to existing mating protocols but could substantially reduce animal wastage, technical time, and cage costs as well as improve animal welfare. By priming females in advance, visually selecting receptive females, and pairing overnight rather than for 3 days, a smaller number of animals would have to be mated, requiring fewer sterile singly housed males. In turn, this would minimize the number of mice suffering the negative impact of isolation on their health and well-being,26 as well as reduce the number of regulated procedures conducted in units using surgically sterilized males.
Experiment 2 considered alternative strategies for inducing estrus using various olfactory and social cues and establishing whether the success rates obtained by exposure to male pheromones could be improved. The results indicated that indirect exposure to a live male and, even more markedly, lactating females, significantly increased receptivity levels, with females spending prolonged periods of time in proestrus/estrus (Figure S1). Significantly, these levels of receptivity induction were greater than with sham soiled bedding, pregnant females, or, more relevantly, soiled bedding, the existing gold standard for induction of the Whitten effect. Receptivity levels were also greater than those achieved with progesterone-based synchronization (63%).18 Although exposure to soiled male bedding has proven sufficient to achieve estrus induction, it is likely that the additional stimulus of social cues such as ultrasonic vocalizations as part of courtship17,30 may have been incremental/synergistic to simple pheromonal stimulation and enabled by the experimental housing setup (i. e., the multiple messages hypothesis). In this regard, male mice exposed to multimodal female ultrasonic vocalizations and urinary pheromonal combinations have analogously been shown to display greater courtship efforts.34 Importantly, the number of females deemed to be in estrus/proestrus in experiment 2 did not reach the high levels obtained in experiment 1. This could be due to the animals in the second experiment only having indirect contact with soiled bedding, which was further admixed with a larger volume of clean bedding, resulting in a diluted and overall weaker pheromonal stimulation.
Our findings with lactating females were very striking in terms of their impact on estrus induction. Work by others15 has demonstrated that naïve females acquire maternal behaviors through olfactory cues within days of living with a litter and have estrogen receptor activity similar to their pregnant counterparts. Moreover, exposure to lactating female urine has been reported to accelerate sexual maturation and receptivity as well as prolong the duration of estrus, putatively because of hormone-dependent urinary pheromone(s) and/or metabolites, which conforms with our experimental findings.11,13,21 In this regard, it is believed that the purpose of this signaling mechanism may be to communicate to naïve females that environmental and/or social conditions are favorable for reproductive activities.11,13 Contrary to previous consistent reports11,13,21; however, we noted no benefits of exposure to pregnant females on estrus induction. This could potentially reflect the different methodologies used (nare painting in the original studies), which could alter the strength of the pheromonal signaling, and the different housing regimens (single housing of females).
In addition to reducing the overall number of females used, the benefits of adopting our experimental strategies for housing breeding/embryo recipient females offer further benefits beyond estrus synchronization, including improved maternal ability, reduced pup mortality, and companionship.15,31 Additionally, this strategy would also reduce the risk of exposing non-receptive females to the attention of a male. While non-receptive females reject advances from males,45 such forced interactions risk causing unnecessary stress and fear through females’ inability to escape unwanted attention. Together with the additional stress of being transferred to a mating cage (i. e., into a new environment),33 existing approaches have a combined, and potentially avoidable, negative impact on females’ welfare, over and above animal wastage and reliance on regulated procedures.
The experimental design used in our work could be optimized for use in animal units. For example, a harem breeding program consisting of up to 4 outbred females housed with a male could be maintained in the treatment side of the GR1800 double-decker Sealsafe Individually Ventilated Cages, providing up to 9 naïve females on the experimental side double exposure to both multimodal male stimulation and lactating females, the 2 stimuli which we found to be the most effective at inducing sexual receptivity. Such an approach could generate large numbers of recipients, reduce the number of males needed for breeding overall, and improve pup survival through inherent cross-fostering opportunities.
There are 2 principal limitations with this study. First, the universality of the results presented herein warrants verification in larger numbers of animals to ensure consistency in estrus induction levels. If the synchronization achieved in experiment 1 could be replicated by exposure to either live males and lactating females (which, of note, was not achieved in experiment 2), it would be possible to synchronize a large number of females for a specific time point (e.g., on day 7 as in experiment 1). Consequently, we consider this to be a foundational study that, ideally, warrants replication across several large facilities to ensure reproducible validation of our reported findings. Second, the levels of female receptivity were assumed based on the interpretation of vaginal cytology rather than being confirmed by mating (which was not possible given the duration of the experiment and its design). If the present findings were validated in larger scale studies, we would anticipate that the need for cytologic assessment could be removed altogether, as this was simply included for confirmatory purposes in this study. In this regard, this could be replaced by visual inspection of the vulva, a skill that animal technicians can easily acquire, given the larger pool of receptive animals available for screening.
In summary, this 2-part study investigated the relative merits of creative, welfare-driven approaches to mouse housing as a non-invasive, physiologic strategy for estrous cycle synchronization in mice. Experiment 1 indicated that Whitten effect-based estrus induction was independent of estrous stage, and that peak receptivity was achieved on days 6 and 7 at the time of the animals’ second cycle rather than their first. Experiment 2 assessed the relative merits of using multimodal sociosexual cues in estrus induction rather than relying on more ‘traditional’ pheromonal stimuli, with indirect exposure of naïve females to either lactating females and stud males being the most effective for increasing receptivity levels and duration, and outperforming gold standard olfaction-based approaches including the Whitten effect. While the experimental housing and synchronization regimens proposed herein require some deviation from conventional housing arrangements, they have the potential to markedly improve animal welfare by reducing the number of recipient females needed and, thus, wastage; minimizing animal manipulation and invasive procedures for both females and males by eliminating the need for injectable agents and vasectomy; reducing stress by reducing repeated restraint/handling and cage transfers; enhancing social interactions of the animals; reducing technician skill requirements; and providing greater environmental enrichment and opportunity to express more natural behaviors (e.g., climbing). While the use of such adapted housing may require an initial financial outlay, it aligns very well with the goal of improved animal welfare.
Supplementary Materials
Figure S1.Population synchronization data from experiment 2. 0 = sexually nonreceptive (metestrus, diestrus); 1 = sexually receptive (proestrus, estrus).
Table S1.Mouse FELASA complete PRIA list of infectious diseases.
Experiment 1 flow chart.
Vaginal cytology samples from the 4 stages of the estrous cycle. Three cell types are identified: leukocytes (circle), cornified epithelial (black arrow), and nucleated epithelial (white arrow). Stages of estrous include (A) proestrus, (B) estrus, (C) metestrus, and (D) diestrus.7
Experiment 2 flow chart.
Representation of receptivity to illustrate synchronization within groups over the 21-day monitoring period (proestrus: n = 10; estrus: n = 3; metestrus: n = 5; and diestrus: n = 9).
Representation of receptivity within pooled animals over the 21-day monitoring period (n = 27) to illustrate synchronization within the population.
Boxplot of days receptive in each treatment group. Groups with shared superscript letters are not statistically significant from each other; those with different superscripts are P < 0.05.
Contributor Notes