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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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, 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.

Introduction

Novel contributions
The rodent estrous cycle 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 "estrous" implies a sense of inferiority and negativity. Euripides used the term to describe a "frenzy" 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—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 hazardous stigma explored in the 'Translation to Humans' section. This study aims to address these three issues in three ways—(A) 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—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 "typical" 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's cycle is unique, and deviations based on animal strain, physiological processes, and environmental conditions are expected. In fact, one of the most "normal" 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' 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—from shelving height and smartphone size to police body armor fitting and missed cancer diagnoses42.

The description of menstruation as unsanitary, destructive, and toxic—seen in revered texts, media, dictionaries, and medical teachings—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 'failure to conceive'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.

Protocol

All handling and procedure methods outlined in this protocol align with National Institutes of Health (NIH) animal care and use guidelines and have been approved by the Institutional Animal Care and Use Committee (IACUC) of Pepperdine University and The UCLA Chancellor's Animal Research Committee (ARC).

1. Animal care and use

  1. Acquire female rats, in numbers according to power analysis, and male rats to promote the Whitten effect or more consistent cycling46. Determine the strain based on the objective of the study in known databases47.
    NOTE: The current data reflect that of female adolescent SD International Genetic Standardization Program (IGS) in the presence of male SD rats located at both Pepperdine University and UCLA laboratories as part of a collaborative study. These rats arrived in separate groups at 28 days of age, and the estrous cycle progression was monitored for either 10 or 20 days to demonstrate differences on acute and chronic levels, beginning at 34 days of age (following a 7-day acclimation period).
  2. Before handling, allow for a quarantine and/or an acclimation period for physiological stabilization following transportation and adjustment to the new environment.
    NOTE: A 3-day minimum period has been cited, with a 7-day period recommended48,49,50,51,52. Overall, this is dependent on the transportation conditions, animal strain, and study objectives.
  3. Ensure that stress is reduced with the use of an acclimation period, as stress can disrupt proper reproductive system functioning53. However, do not overcompensate by attempting to eliminate it, as a moderate amount of stress is beneficial to the animals' well being51.
  4. Host rats in a temperature- (68-79 °F, i.e., 20-26 °C) and humidity-controlled (30-70%) environment by contacting the vivarium or laboratory managers and ensuring these features. Distribute water and chow ad libitum with nutrient components listed on the company website and cage cleaning once a week.
    NOTE: In this study, the rats were housed in groups of 2 separated by sex in 19" x 10" x 8" clear reusable plastic cages and had access to corncob bedding that was changed once a week. The temperature was maintained at 70 °F and humidity at 35-79%, with an average of 62%.
  5. Check for the development of ringtail or ischemic necrosis of the tail and toes for evidence of low relative humidity levels and extreme temperatures, which may cause the alternation of biological responses.
    NOTE: Temperature and humidity are important for the reproductive system, sexual maturation, and estrous cyclicity54,55,56,57,58.
  6. Ensure proper and balanced illumination throughout the housing space by depositing equal amounts of light sources throughout the laboratory space that act on a time-controlled light:dark system.
    NOTE: Here, a 12:12-h light:dark cycle, with lights on from 06:00-18:00 h, was controlled by 2,550-lumen Linear LED bulbs.
  7. Follow lux requirements provided52 based on variations of animals' pigmentation, age, strain, sex, and hormonal status.
    ​NOTE: When researchers categorize the cell samples collected, consistent lighting will allow for proper visual detection and reliable staging59. The duration and intensity of light are directly related to the reproductive system, sexual maturation, and estrous cycling54,55,56,57,60,61.

2. Equipment and experiment preparation

  1. Review the categorizing determinants (seen in Figure 2A): how to identify each stage of the estrous cycle and how to operate the microscope and camera equipment.
  2. Ensure that each subject to be monitored has reached sexual maturation and shows appropriate indicators of development—VO, body weight, and age. Weigh the rats and examine them for VO between at the same time each day for accurate comparisons and transfer them with an approved handling method. Consult the university-affiliated veterinarian if an animal loses more than 20% of its previous body weight.
    NOTE: The VO remains caudal to the urethral opening and cranial to the anus, located between the two, as depicted in Figure 3.
  3. As these factors are strain-dependent, check with the supplier for specifications and consider environmental factors specific to the laboratory62.
    NOTE: In general, this will occur between 32-34 days of age and an average body weight ranging from 75-150 grams63 for SD rats and is indicated by a circular-shaped opening previously covered by a membranous sheath.
  4. Select a sample collection period appropriate for the group of rats being monitored to prevent collecting transitional samples. First, sample a few animals at 2 or 3 different timepoints throughout the day to determine the time where most cycle stages are present (e.g., sampling at 12:00 hours, 13:00 hours, and 14:00 hours for different animals). Complete the vaginal lavage at the same time each day for consistent and reliable staging.
    NOTE: It has been reported that the hours between 12:00 and 14:00 h are best for capturing all stages. In this study, estrous cycle monitoring occurred between 12:00 and 14:00 h, handled with the compression-style hold (see step 3.4). The importance of estrous cycle monitoring timing relative to other experimental interventions (e.g., behavioral conditioning, medication) is a developing area of research and can be further explored11. Determining the duration of the estrous cycle monitoring is study-dependent and can be further explored in published studies11,33.
  5. Remove the protective cover from the microscope and attach the camera to the computer by removing the protective camera lens cover and placing the lens over the eyepiece of the microscope.
  6. Then, open the preselected software on the computer. To use the software selected in this study, select the camera attached to the USB located on the left-hand side of the screen, under the tab labeled Camera List. Ensure that the USB camera is properly connected to the computer, which will read No Device under the tab labeled Camera List, if not.
  7. Once the USB camera has been selected under the tab, turn on the microscope light switch located on the base.
  8. Create a folder on the computer designated for the cell sample photos. Create a file folder for each separate day that data are collected, preprepared before images are taken.
  9. With the equipment prepared, retrieve the subject's cage from its holding place and bring it to the sample collection station.

3. Collection of vaginal cells

  1. Retrieve a disposable syringe and fill each of the syringes with 0.2 mL of sterile 0.9% NaCl. If air bubbles are present, gently flick the barrel syringe until all the air bubbles have reached the open tip of the syringe and expel the air. If there are still air bubbles present, expel the solution back into the NaCl receptacle and refill until there are none.
    NOTE: Excessive flicking may result in the formation of more air bubbles.
  2. Return each syringe to the plastic wrapping to maintain a sterile field, with the tip of the syringe inside the sealed portion of the wrapping.
  3. Open the cage and gently lift the subject by either the base of the tail or the trunk of the body, closing the lid of the cage to prevent others from exiting. Select a holding method from those listed below based on personal preference and animal response.
  4. Use the compression-style hold for adolescent rats by placing the subject against the upper chest region, with the subject's nose pointing down at the ground. Before beginning the swab, ensure that the subject is compressed enough to prevent movement but is comfortable and safe in the hold. Expose the vaginal canal of the subject by gently flexing the tail before inserting the syringe.
  5. Use the Hind Leg Lift for adult rats by placing the animal's forepaws on either the top or side of the cage, while the tail and hindlimbs are restrained with a gentle hold between the first and second fingers, leaving the thumb free to operate the syringe64.
  6. Allow for the animals to acclimate to the handling and monitoring. Handle the animals gently yet securely to reduce excess stress and to protect the researcher from aggression such as biting.
    NOTE: The first few days of monitoring may not produce the desired results as the animals acclimate to their conditions. Handling the animals to collect body weights during the acclimation period can assist this transition33.
  7. While holding the syringe steady with the forefinger and middle finger, insert the tip of the syringe (no more than 2 mm) at an angle parallel to the vaginal canal. Slowly expel the NaCl into the canal by pushing the plunger inwards. Do not insert the syringe further into the canal, as doing so may disrupt the estrous cycle.
  8. Extract the NaCl from the vaginal canal by pulling the plunger of the syringe away from the epithelial lining (upwards). If there is difficulty keeping the subject in the hold during this process, place them back into the cage for a short rest period before attempting NaCl extraction.
  9. Once the cell sample has been collected, place the subject back in the cage and repeat this procedure for each animal before all samples are evaluated under the microscope.
    ​NOTE: Alternatively, each sample can be collected and evaluated before moving on to the next animal. An animal may require a second lavage if the sample cannot be categorized. The same syringe from the initial collection can be reused if it does not contact the saline directly in the container and only for the same animal.

4. Sample evaluation

  1. Begin the categorization by examining the vaginal fluid sample extracted. Record the viscosity as either viscous or nonviscous and the coloration as either opaque or transparent on the description document or other recording system.
    NOTE: This section of the protocol can be performed at the time of sample collection or later.
  2. Expel 2-3 drops of the fluid onto a microscope slide and place a microscope cover glass on top of the slide. Place the cover glass on the microscope slide from the top of the slide to the bottom or from one side of the slide to the other to prevent the formation of air bubbles. If possible, leave approximately half of the collected sample in the syringe if further examination is required and to prevent an excess amount of fluid from being placed on the slide.
  3. Locate the cells collected by moving the microscope slide across the stage. If there are too few cells or a high amount of debris, expel the remaining fluid onto a new slide and reexamine. If the amount of sample left in the syringe is insufficient, or if the second drop is presenting similar issues, collect another sample from the subject before attempting to identify the estrous cycle stage.
  4. Once the cells have been located and before touching the computer or the computer keyboard, remove the one glove to prevent soiling the keyboard.
  5. Acquire images of the cell samples by clicking on the function labeled Snap on the left-hand side of the software panel.
  6. Then, save the file by clicking on Save as under the File icon on the top left corner of the page. Save the photo under a prelabeled folder on the computer.
    NOTE: Example label template: #subjectnumber_date collected_estrous stage_objective lens used.
  7. Take more than one photo at each objective lens if there are not many cells within each frame.
    NOTE: Example labels for multiple images: #1_01/09/2021_EST_4x1 and #1_01/09/2021_EST_4x2.
  8. Repeat the procedure for each collected sample under multiple objective lenses. Include at least one smaller objectification, such as 4x, and at least one larger objectification, such as 20x.
  9. Upload the images to a shared drive/folder or an external hard drive so all researchers involved have access to the files and there are backup copies available.

5. Stage categorization

  1. Set up the computer screen to simultaneously view the photos taken and the recording sheet (Figure 4A-C).
    NOTE: This will allow for the documentation to occur while viewing the sample collected. This portion of the protocol can be completed at the time of sample collection or later.
  2. Determine which cell types are present in the sample. Select from the four options listed in steps 5.2.1-5.2.4 using the criteria and record the findings.
    1. Anucleated keratinized epithelial (AKE)/cornified epithelial cells
      1. Look for jagged or angular-edged cells, as seen in Figure 2B and Figure 5C, which despite the lack of nuclei, may show light round areas (nuclear ghosts) within the cell that represent where a nucleus was once present. Use higher magnification, such as 20x and above, to differentiate between such nucleated and anucleated cells.
      2. Use higher magnification to distinguish the keratinized or cornified portion of the cell—a thin layer of cells lacking nuclei and filled with keratin—if desired.
        NOTE: In addition to their jagged appearance, these can also be distinguished by how they can fold or disassemble, creating jagged and elongated structures known as keratin bars.
    2. Large nucleated epithelial (LNE) cells
      1. Look for these typically round- to polygonal-shaped cells encased by irregular, jagged, or angular borders.
      2. Observe how their nuclei may take on various forms, ranging from intact to degenerate or pykontic, relating to the irreversible condensation of chromatin in the nucleus of a cell undergoing death or deterioration, as seen in Figure 2B and Figure 5D1,2. Take note of how these nuclei occupy less space than the cytoplasm within the cell, with a lower nuclear to cytoplasmic (N:C) ratio than the small epithelial cells. Look out for cytoplasmic granules that can be seen at higher magnifications13.
    3. Leukocytes (LEUs)/neutrophils/polymorphonuclear cells
      1. Look for these compact, spherical cells with multilobulated nuclei (hence, known as polymorphonuclear cells), which vanish as the cell matures (Figure 2B and Figure 5A). Higher magnification (e.g., 40x) can be used to observe the multilobulated nuclei.
        NOTE: Upon collection and preparation, these cells may condense, fold, or rupture.
    4. Small nucleated epithelial (SNE) cells
      1. Look out for these round- to oval-shaped cells that are larger than the neutrophils described above.
      2. Observe the round nuclei of these nonkeratinized epithelial cells (Figure 2B), which take up a larger amount of space than the cytoplasm inside the cell, creating a higher N:C ratio relative to large epithelial cells.
        NOTE: Upon collection and preparation, these cells may fold or overlap to create a shape that resembles a string or bar, as demonstrated in Figure 5B1.
  3. Examine how the cells present in the sample are organized for each objectification. Use the lower objectification, such as 4x, to view a representative view of the overall cell arrangement. Record whether the cells are clumped together (C), evenly dispersed (ED), or randomly dispersed (RD) (see Figure 4C), and note the specific organization of each cell type (e.g., small nucleated epithelial cells are clumped, and neutrophils are evenly distributed).
  4. Next, visually estimate and record the total cell quantity (a smidge, moderate, numerous) and the individual cell quantities (percentage of each cell type present).
    NOTE: A smidge represents the smallest number of cells present that can be utilized to determine the sample categorization, numerous represents the presence of a countless number of cells that either account for most if not all of the space on the slide or are stacked on top of one another, and a moderate number of cells represents a comparatively average number of cells (examples seen in Figure 5A-D and Figure 6A-D).
  5. Note whether there are any deviations from either the listed criteria or aspects that are typical for the specific subject in the 'abnormalities' category and consult with a veterinarian if needed.
  6. Determine which estrous cycle stage is being presented in the sample utilizing the categorizing components and the descriptions below.
    1. DIE
      1. Look for LEUs as the dominant or only cell type present, arranged in a clumped manner at the beginning of DIE but more dispersed in late stages.
        NOTE: While transitioning into DIE, the quantity of cells may decrease as the epithelial cells begin to break down, as seen in Figure 6D1. At the same time, the number of LEUs begins to increase, and they tend to be arranged in a clumped manner initially and disperse over time.
      2. Note that the total quantity of cells may be comparatively low, most often in the later stages of the DIE period, on the second or third day.
      3. Observe the high amount of mucus that may be present in this stage, which presents as concentrated strands of LEUs (Figure 5A1). Look out for small clumps or cellular strands of SNE cells accompanying the LEUs during late phases in the transition to PRO (Figure 5A1,2).
      4. Observe the viscous and opaque appearance of the vaginal fluid when transitioning into, fully transitioned into, and transitioning out of DIE.
        NOTE: The average duration of this stage is 48 h during a 4-day cycle and possibly 72 h during a 5-day cycle.
    2. PRO
      1. Look for SNE cells as the dominant cells and for LEUs, LNE, and/or AKE cells that can be seen in low numbers. Use high objectification to observe the granular appearance of the SNE cells that are typically arranged in clusters, sheets, or strands during this stage (Figure 5B1,2).
      2. Observe the viscous and opaque appearance of the vaginal fluid when moving from DIE into PRO, and how it becomes nonviscous and transparent once fully transitioned into the PRO stage (average duration of 14 h in rats).
    3. EST
      1. Look for dominance of AKE cells, a diminishment of the SNE cells in EST, and an increase in the number and size of cells as EST continues11,13.
      2. Make a note of the distinguishing feature of the often clustered arrangement of AKE cells, in the form of keratin bars or containing ghost nuclei, which can become more randomly dispersed in the transition from PRO (Figure 6B) and to MET (Figure 6C).
      3. Observe the characteristic nonviscous and transparent vaginal fluid, which can be expected as the rats are transitioning into, fully transitioned into, and transitioned out of EST.
        NOTE: The progression of EST includes much diversification (Figure 5C and Figure 6B, C). The stage typically occurs for an average of 24 h in a 4-day cycle or possibly 48 h in a 5-day cycle.
    4. MET
      1. Look for higher numbers of SNE and LNE cells as the rat is transitioning into MET, either dominant in terms of cell proportion within the canal or close to equal proportion to the LEUs11,13. Further, make a note of the greater amount of debris in MET and the transitions than in other stages due to the epithelial cell decay following EST and moving into DIE.
      2. Observe the lack of consistent arrangement as all cell types are seen and in various amounts (Figure 5D1-3). However, look for the LEUs that are packed or clumped in proximity to the epithelial cells in the beginning stages that may return to the clumped arrangement when transitioning into DIE.
      3. Observe the nonviscous and transparent appearance of the vaginal fluid in this stage and the change to a more viscous and opaque appearance while moving into DIE.
        NOTE: The average duration of this stage is 6-8 h.
  7. Label samples in transitions with the stage the subject is moving towards, with the transition in parenthesis to track when these are collected. Further information on how to distinguish between these 4 stages and their transitions can be found in the Representative Results.
    NOTE: As the samples collected are static and the cycle is dynamic, the slides may depict transitions between stages (seen in Figure 6A-D).
  8. Complete this process for each animal until the monitoring phase is complete.
  9. On either day 11 (45 days of age) or 21 (55 days of age), euthanize the rats with 5% isoflurane and 2% oxygen before a guillotine decapitation. These timepoints may vary depending on the nature of the study.

Results

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...

Discussion

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...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

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

Materials

NameCompanyCatalog NumberComments
AmScope 40X-1000X LED Student Microscope + 5MP USB CameraAmScopePart Number: M150C-E5 EAN: 0608729747796 Model Number: M150C-E5https://www.amazon.com/AmScope-40X-1000X-Student-Microscope-Camera/dp/B00O9GNOTA/ref=sr_1_15?crid=2W9CHTG8YSOTV&
keywords=usb+camera+for+microscope&
qid=1572477663&s=industrial
&sprefix=USB+camera+for+micr%2Cindustrial%2C177&sr=1-15
BD PrecisionGlide Needle Pack, 20G x 1, Short BevelFischer Scientific14-815-526https://www.fishersci.com/shop/products/bd-precisionglide-single-use-needles-short-bevel-regular-wall-4/14815526#?keyword=BD%20PrecisionGlide%20Needle%20Pack,%2020G%20x%201
Bed O Cob 1/8NEWCO93009https://andersonslabbedding.com/cob-products/bed-ocobs-8b/
Corning™ Plain Microscope Slides Plain water-white glassFischer Scientific12-553-7Ahttps://www.fishersci.com/shop/products/corning-plain-microscope-slides-microscope-slides-75-x-25mm/125537a
Corning™ Rectangular Cover GlassesFischer Scientific12-553-464https://www.fishersci.com/shop/products/corning-square-rectangular-cover-glasses-rectangle-no-1-thickness-0-13-0-17mm-size-24-x-50mm/12553464#?keyword=true
Kimberly-Clark Professional™ Kimtech Science™ Kimwipes™ Delicate Task Wipers, 1-PlyFischer Scientific06-666https://www.fishersci.com/shop/products/kimberly-clark-kimtech-science-kimwipes-delicate-task-wipers-7/p-211240?crossRef=kimwipes
Labdiet Rodent Lab Chow 50lb, 15001 NEWCO Specialty and LabDiet5012https://www.labdiet.com/products/standarddiets/rodents/index.html
Linear LED Bulb, UL Type A, T8, Medium Bi-Pin (G13), 4,000 K Color Temperature, Lumens 2550 lmGrainger36UX10https://www.grainger.com/product/36UX10?gclid=CjwKCAjw_
LL2BRAkEiwAv2Y3SW1WdNdkf7
zdIxoT9R6n2DGnrToJHjv-pwCTca4ahQyExrrtWvbgwRoCi4
cQAvD_BwE&s_kwcid=AL!2966!3!335676016696
!p!!g!!led18et8%2F4%2F840&ef_id=
CjwKCAjw_LL2BRAkEiwAv2Y3SW
1WdNdkf7zdIxoT9R6n2DGnrToJ
Hjv-pwCTca4ahQyExrrtWvbgwRo
Ci4cQAvD_BwE:G:s&s_kwcid=AL!2966!3!335676016696!p!!g!!led18et8%2F4%2F840&cm_mmc=
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