This study was conducted through an NIH-funded collaboration between the University of California Los Angeles Brain Injury Research Center (BIRC).

The authors have no conflicts of interest to disclose.

Key steps and important considerations 
Certain critical steps in the provided protocol require emphasis, especially within the collection of vaginal cells. During the vaginal fluid extraction, ensuring the proper angle and depth of syringe insertion is key to producing satisfactory results and ultimately preventing irritation, injury, or cervical stimulation to the animal. The stimulation of the cervix can be one source of pseudopregnancy induction, indicated by 12-14 days of a leukocyte-only vagin...

Novel contributions 
The rodent estrous cycle&#160;has been identified as an essential indicator of wellness. However, unconscious biases of investigators and inaccurate interpretations regarding the female body hinder the scientific community. The very etymology of the word &#34;estrous&#34; implies&#160;a sense of inferiority and negativity. Euripides used the term to describe a &#34;frenzy&#34; or madness, Homer to describe panic, and Plato to describe an irrational drive. This study highlights how these primeval perspectives influence the current scientific community and addresses these concerns through a novel mosaic paradigm&#8212;an updated combination of previously studied methods, expanded in scope for a more comprehensive approach.
The study and use of this technique are necessary, first, as there is no standardized and comprehensive monitoring technique, and data interpretation practices can be unclear. Second, although estrous cycle characteristics are dependent on individual rats being studied, they are often universalized. Third, while hormonal cycles are routine and beneficial processes, they are surrounded by&#160;hazardous stigma explored in the &#39;Translation to Humans&#39; section. This study aims to address these three issues in three ways&#8212;(A)&#160;by describing an in-depth estrous cycle monitoring technique and clarifying how the results can be interpreted, (B) by outlining methods that maintain the integrity and individuality of each cycle, and (C) by calling attention to misconceptions that perpetuate unsubstantiated practices.
This study is also unique in its focus on adolescent rats, a period marked by crucial developmental changes that shed light on various behavioral, anatomical, and physiological manifestations in adulthood1. Building a standardized experimental design to monitor hormonal cycles in an under-researched population while deconstructing common biases will allow for the development of reliable and valid hormonal correlations2,3,4 and the determination of condition-dependent cycle disruptions5,6,7,8,9,10. Ultimately, these novelties serve to expand diagnostic criteria, treatments, and interventions of various wellness concerns.
Fundamental definitions and uses 
The estrous cycle is a collection of dynamic physiological processes that occur in response to the three oscillating female sex steroid hormones: estradiol, leuteinizing hormone (LH), and progesterone (Figure 1A,B). Interactions between the endocrine and central nervous system regulate the cycle, which most often persists for 4-5 days and recurs from the onset of sexual maturation until reproductive senescence and/or cessation. It is divided into separate categories based on hormone levels&#8212;most commonly into the 4 stages of diestrus (DIE), proestrus (PRO), estrus (EST), and metestrus (MET), which progress in a circular fashion. The number of divisions can range from 3 stages11 to 13 stages12, depending on the nature of the study13. The lower number of divisions often excludes MET as a stage and classifies it as a short-duration transitionary period. The higher number typically includes subsections that allow for a closer inspection of phenomena such as tumor development or spontaneous pseudopregnancy, the physiological state of pregnancy without embryonic implantation12,14,15.
In this study, the stages were identified through components of the vaginal canal, named the 3 categorizing determinants-cell type(s) present, cell arrangement, and cell quantity (Figure 2A-D). While the condition of the vaginal fluid was not monitored in this study, it is recommended to include it as a fourth categorizing component. Further information on examining the vaginal fluid can be found in the reference list16. The categorizing components can be examined by extracting cells via vaginal lavage, the primary technique recommended in modern-day estrous cycle monitoring. While the in-depth physiological processes within each stage are outside the scope of this study, more information can be found in the literature17.
The use and continued development of this estrous cycle monitoring technique is rooted in the connections between sex steroid hormones and the function of bodily systems such as the cardiovascular system18, endocrine system8, and central nervous system19,20,21. At the same time, estrous cycle monitoring may not always be necessary when female rodents are involved22,23,24,25. Rather, it is important first to consider if sex differences have been reported in the specific area of study, which can be further explored in published reviews22,23. Though estrous cycle monitoring is vital in a broad spectrum of research investigations, it should not be seen as an obstacle to including female rodents in experiments. While this technique may appear complex and time-consuming, the procedure itself can take less than 15 min to complete, depending on the investigator, and is cost-effective. Overall, the inclusion of female rodents in scientific studies is advantageous to the understanding of bodily systems, various conditions and pathologies, and general wellness, as these developments have been mainly based on the male body template.
Universal parameters and natural variabilities in the rodent 
Establishing ranges for aspects seen as &#34;typical&#34; is necessary to define standard cycle patterns, set parameters for comparative and analytic purposes, and detecting abnormalities and outliers. At the same time, it is also important to recognize that each rat&#39;s cycle is unique, and deviations based on animal strain, physiological processes, and environmental conditions are expected. In fact, one of the most &#34;normal&#34; aspects of the estrous cycle is variability. This is seen in the total cycle length, with a range of 3-38 days26,27; the age of sexual maturation that can range from 32-34 days to multiple weeks28,29,30; what is considered acyclical11, and the categorizing determinant patterns11,13. Overall, there is no universal template for the estrous cycle, and translating that to both the scientific community and the general public is an important part of the experimental process.
Experimental timepoints and developmental age 
Recognizing this principle of variability assists in building a reliable and valid experimental design. For example, the start of estrous cycling monitoring relies on the rats&#39; anatomical and physiological development, which varies based on environmental and physiological factors. Monitoring cannot begin until the development of the vaginal opening (VO), which is the external vaginal orifice surrounded by the vulva that leads to the interior portion of the vaginal canal (Figure 3A-D). While the VO often fully develops between the ages of 32 and 34 days, it remains individualized to each subject, and much about the process remains unknown. This opening has been used to identify the onset of sexual maturation, which has been linked to the increase of estradiol31, the maturation of the hypothalamic-pituitary-ovarian axis32, and the first ovulation in rats17, 33,34,35. However, recent publications have found that it is only an indirect marker of reproductive development, as it can become uncoupled from hormonal and developmental occurrences in unfavorable environments31 and may represent changes in estradiol levels rather than sexual maturation33. Therefore, it is recommended to not rely solely on the VO to determine developmental age and as a qualifier for estrous cycle monitoring36 but to also utilize the appearance of the first EST stage and cornification of the epithelial cells30 to mark the onset of sexual maturation.
Body weight is notably correlated with developmental age during the adolescent period in rodents30,37 and can therefore also assist in determining developmental age in this period. Proposed mechanisms related to this phenomenon include the stimulation of hormones necessary for reproductive development, such as growth hormone, and the inhibition of the hypothalamic-pituitary adrenal (HPA) axis by the appetite regulator, leptin30. However, it is not recommended to utilize this measure as the sole indicator of developmental age due to the large variance seen between rats across species and vendor providers38. The variability seen in the development of the VO and body weight exemplify the importance of the concept in the overall experimental process.
Translation to humans: cultural and scientific contexts 
The translational relationship of animal-to-human reproductive studies is bidirectional. The results from animal-based studies influence how the human processes are assessed, approached, and analyzed39. The perception of the human reproductive system and its related processes influence how animals are studied. In fact, one of the loudest indications for further research in this area stems from biased sociocultural beliefs related to hormonal cycles that influence the scientific process. Many of these conventions are derived from a general cultural aversion to discussing menstruation, which has led to a data gap in well-substantiated knowledge40,41. This has a spectrum of consequences that span from minor to lethal&#8212;from shelving height and smartphone size to police body armor fitting and missed cancer diagnoses42.
The description of menstruation as unsanitary, destructive, and toxic&#8212;seen in revered texts, media, dictionaries, and medical teachings&#8212;is conserved by scientific publications. This occurs through inaccurate and biased descriptions of hormonal cycles, the isolation of the reproductive system from its neuroendocrine counterparts and environmental influences, and the reductionist perspective of the completion of a cycle as a &#39;failure to conceive&#39;43,44. This leads to the creation of unsound experimental practices, such as the omission of external variables that influence hormonal cycles, determining start and endpoints based solely on anatomical developments, and measuring cycle advancement in a linear rather than circular fashion. Despite the direct correlation between sociocultural factors and biological consequences, it is not often considered in scientific literature. Through the inspection of more holistic publications43,44,45, researchers can deconstruct these stigmas and create more reliable and valid experimental designs.

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rodent estrous cycle monitoring utilizing vaginal lavage: no such thing as a normal cycle

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley (SD) adolescent rats. This study demonstrates the complexity of hormonal cycles and the broad spectrum of understanding required to construct a reliable and valid monitoring technique. Through an in-depth examination of principal experimental design and procedural elements,&#160;this description of the cycle and its fundamental principles provides a framework for further understanding and deconstructs misconceptions for future replication.
Along with an outline of the sample collection process employing vaginal lavage, the procedure describes the mechanism of data categorization into the four-stage model of proestrus, estrus, metestrus, and diestrus. These stages are characterized by a new proposed approach, utilizing the 4 categorizing determinants of vaginal fluid condition, cell type(s) present, cell arrangement, and cell quantity at the time of collection. Variations of each stage, favorable and unfavorable samples, the distinction between cyclicity and acyclicity, and graphic depictions of the collected categorizing components are presented alongside effective interpretive and organizational practices of the data. Overall, these tools allow for the publication of quantifiable data ranges for the first time, leading to the standardization of categorization factors upon replication.

The current data reflect that of female adolescent SD International Genetic Standardization Program (IGS) in the presence of male SD rats. These animals were located at both Pepperdine University and UCLA laboratories as part of a collaborative study. Figure 5 presents multiple variations of the 4 cycle stages. Figure 5A1 was identified as a diestrus sample with several cell types present. This example demonstrates that samples with a larger num...

Watch this Scientific Journal Video about Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle at JoVE.com

Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle

This study details the crucial factors to consider in experimental designs involving female rats. In a larger sense, these data serve to decrease stigma and assist in the development of more inclusive diagnostic and intervention tools.

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley ...

rodent-estrous-cycle-monitoring-utilizing-vaginal-lavage-no-such

Research

JoVE Journal

Biology

7.4K Views.  UCLA, David Geffen School of Medicine. This study details the crucial factors to consider in experimental designs involving female rats. In a larger sense, these data serve to decrease stigma and assist in the development of more inclusive diagnostic and intervention tools. 

Video: Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Behavior

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

Assessment of Stress Effects on Cognitive Flexibility using an Operant Strategy Shifting Paradigm

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and duration of the stressor; 2) type of cognitive function under study; and 3) timing of the stressor in relation to learning or executing the cognitive task. Furthermore, sex differences among the effects of stress on cognitive function have been widely documented. Described here is an adaptation of an automated operant strategy shifting paradigm to assess how variations in stress affect cognitive flexibility in male and female Sprague Dawley rats. Specifically, restraint stress is used before or after training in this operant-based task to examine how stress affects cognitive performance in both sexes. Particular brain areas associated with each task in this automated paradigm have been well-established (i.e., the medial prefrontal cortex and orbitofrontal cortex). This allows for targeted manipulations during the experiment or the assessment of particular genes and proteins in these regions upon completion of the paradigm. This paradigm also allows for the detection of different types of performance errors that occur after stress, each of which has defined neural substrates. Also identified are distinct sex differences in perseverative errors after a repeated restraint stress paradigm. The use of these techniques in a preclinical model may reveal how stress affects the brain and impairs cognition in psychiatric disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), which display marked sex differences in prevalence.

Stressful life events impair cognitive function, increasing the risk of psychiatric disorders. This protocol illustrates how stress affects cognitive flexibility using an automated operant strategy shifting paradigm in male and female Sprague Dawley rats. Specific brain areas underlying particular behaviors are discussed, and translational relevance of results are explored.

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Medicine

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Immunology and Infection

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Neuroscience

DREADDs Technology and Automated Blood Sampling

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...