Name: rodent estrous cycle monitoring utilizing vaginal lavage: no such thing as a normal cycle
Uploaded: 2021-08-30T15:00:00
Description: Watch this Scientific Journal Video about Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle at JoVE.com

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

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&#39;s Animal Research Committee (ARC).
1. Animal care and use
<ol>
	<li>Acquire female rats, in numbers according to power analysis, and male rats to promote the Whitten effect or more consistent cycling46</s...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AmScope 40X-1000X LED Student Microscope + 5MP USB Camera</td>
			<td>AmScope</td>
			<td>Part Number: M150C-E5 EAN: 0608729747796 Model Number: M150C-E5</td>
			<td>https://www.amazon.com/AmScope-40X-1000X-Student-Microscope-Camera/dp/B00O9GNOTA/ref=sr_1_15?crid=2W9CHTG8YSOTV&#38;amp; 
			keywords=usb+camera+for+microscope&#38;amp; 
			qid=1572477663&#38;amp;s=industrial 
			&#38;amp;sprefix=USB+camera+for+micr%2Cindustrial%2C177&#38;amp;sr=1-15</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle Pack, 20G x 1, Short Bevel</td>
			<td>Fischer Scientific</td>
			<td>14-815-526</td>
			<td>https://www.fishersci.com/shop/products/bd-precisionglide-single-use-needles-short-bevel-regular-wall-4/14815526#?keyword=BD%20PrecisionGlide%20Needle%20Pack,%2020G%20x%201</td>
		</tr>
		<tr>
			<td>Bed O Cob 1/8</td>
			<td>NEWCO</td>
			<td>93009</td>
			<td>https://andersonslabbedding.com/cob-products/bed-ocobs-8b/</td>
		</tr>
		<tr>
			<td>Corning&#8482; Plain Microscope Slides Plain water-white glass</td>
			<td>Fischer Scientific</td>
			<td>12-553-7A</td>
			<td>https://www.fishersci.com/shop/products/corning-plain-microscope-slides-microscope-slides-75-x-25mm/125537a</td>
		</tr>
		<tr>
			<td>Corning&#8482; Rectangular Cover Glasses</td>
			<td>Fischer Scientific</td>
			<td>12-553-464</td>
			<td>https://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</td>
		</tr>
		<tr>
			<td>Kimberly-Clark Professional&#8482; Kimtech Science&#8482; Kimwipes&#8482; Delicate Task Wipers, 1-Ply</td>
			<td>Fischer Scientific</td>
			<td>06-666</td>
			<td>https://www.fishersci.com/shop/products/kimberly-clark-kimtech-science-kimwipes-delicate-task-wipers-7/p-211240?crossRef=kimwipes</td>
		</tr>
		<tr>
			<td>Labdiet Rodent Lab Chow 50lb, 15001&#160;</td>
			<td>NEWCO Specialty and LabDiet</td>
			<td>5012</td>
			<td>https://www.labdiet.com/products/standarddiets/rodents/index.html</td>
		</tr>
		<tr>
			<td>Linear LED Bulb, UL Type A, T8, Medium Bi-Pin (G13), 4,000 K Color Temperature, Lumens 2550 lm</td>
			<td>Grainger</td>
			<td>36UX10</td>
			<td>https://www.grainger.com/product/36UX10?gclid=CjwKCAjw_ 
			LL2BRAkEiwAv2Y3SW1WdNdkf7 
			zdIxoT9R6n2DGnrToJHjv-pwCTca4ahQyExrrtWvbgwRoCi4 
			cQAvD_BwE&#38;s_kwcid=AL!2966!3!335676016696 
			!p!!g!!led18et8%2F4%2F840&#38;ef_id= 
			CjwKCAjw_LL2BRAkEiwAv2Y3SW 
			1WdNdkf7zdIxoT9R6n2DGnrToJ 
			Hjv-pwCTca4ahQyExrrtWvbgwRo 
			Ci4cQAvD_BwE:G:s&#38;s_kwcid=AL!2966!3!335676016696!p!!g!!led18et8%2F4%2F840&#38;cm_mmc= 
			PPC:+Google+PPC</td>
		</tr>
		<tr>
			<td>Sodium Chloride Injection Bags, 0.9%</td>
			<td>Live Action Safety</td>
			<td>ABB079830939</td>
			<td>https://www.liveactionsafety.com/injection-iv-solution-9-sodium-chloride-1000ml-bags/</td>
		</tr>
		<tr>
			<td>Syringe Sterile 1ml&#160; with Luer Slip Tip - 100 Syringes by BH Supplies</td>
			<td>BH Supplies</td>
			<td>ASIN: B07BQDRDC2 UPC: 638632928821</td>
			<td>https://www.amazon.com/1ml-Syringe-Sterile-Luer-Slip/dp/B07BQDRDC2/ref=sr_1_1_sspa?crid=13S8EGEUK90G7&#38;amp; 
			keywords=1ml+sterile+syringe&#38;amp;qid= 
			1572478649&#38;amp;s=industrial 
			&#38;amp;sprefix=1+ml+steri%2Cindustrial%2C187&#38;amp;sr=1-1-spons&#38;amp;psc=1&#38;amp;spLa= 
			ZW5jcnlwdGVkUXVhbGlmaWVy 
			PUEyRlo4NFdZWkJLWkxGJm 
			VuY3J5cHRlZElkPUEwMDEzODQ 
			yMjNWNzdWM0hTNzVBRCZlbmNy 
			eXB0ZWRBZElkPUEwNDI3NzAzM 
			0E5SzVKMkxaQVc2JndpZGdldE5h 
			bWU9c3BfYXRmJmFjdGlvbj1jbGlja 
			1JlZGlyZWN0JmRvTm90TG9nQ2 
			xpY2s9dHJ1ZQ==</td>
		</tr>
		<tr>
			<td>Wire lids and floors Mouse Maternity Wire Bar LidUsed with Rat Cage (10&#34; X 19&#34; x 8&#34;H )Overall dimen</td>
			<td>Allentown</td>
			<td>LV40326013</td>
			<td>https://www.labx.com/item/wire-lids-and-floors-mouse-maternity-wire-bar-lidused/LV40326013#description</td>
		</tr>
		<tr>
			<td>Ultra Lightweight Tissue and Plastic 17&#39; x 24&#39; Disposable Underpad</td>
			<td>Medline</td>
			<td>EAN: 0480196288558 
			&#160;Global Trade Identification Number: 40080196288558</td>
			<td>https://www.amazon.com/Medline-Industries-MSC281224C-Lightweight-Disposable/dp/B00A2G67YU/ref=sr_1_4?keywords=medline+industries+surgical+pads&#38;amp;qid=1572475853&#38;amp; 
			sr=8-4</td>
		</tr>
	</tbody>
</table>

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 ...

Research

JoVE Journal

Biology

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

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 ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Application of Genetically Encoded Fluorescent Nitric Oxide NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Probes, the geNOps, for Real-time Imaging of NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...