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

Robert, Hannah; Ferguson, Lindsay; Reins, Olivia; Greco, Tiffany; Prins, Mayumi L.; Folkerts, Michael

doi:10.3791/62884

Cited by 5 publications

(10 citation statements)

References 55 publications

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“…We demonstrate that the method outperforms traditional staging in detecting behavioral effects, and correlation with uterine histology confirms its greater physiological relevance. Our data counter perceptions that tracking the cycle requires expert knowledge and that irregular cycles are common 15,16,27 .…”

Section: Discussioncontrasting

confidence: 78%

“…Timing by the cornified peak alone, we found that the vast majority of cycles lasted four days and that almost all rats had regular cycles. This counters the common perception that cycles are often irregular, or, as stated in the title of a recent guide to staging 15 , that there is “no such thing as a normal cycle.” If a normal cycle requires each of the traditional stages to appear in order or for consistent durations, most of our rats would be considered irregular. Since it is common to exclude subjects without regular cycles and there is no agreed-upon definition of regular, differences in exclusion criteria could explain some conflicting results in the literature.…”

Section: Discussionmentioning

confidence: 73%

“…Disagreement between studies can fuel the perception that the estrous cycle is an uncontrollable confound, but it may actually be driven by a lack of universal standards for tracking and reporting the cycle 13 . Conventional methods of tracking the estrous cycle are complex and require expert knowledge [15][16][17] , so a straightforward, standardized approach tailored for behavioral research is necessary to improve reproducibility between studies and lower a barrier to the use of female subjects.…”

Section: Introductionmentioning

confidence: 99%

“…Traditional tracking methods assign vaginal smears to one of four classic stages of mammalian reproduction: estrus, metestrus, diestrus, and proestrus [22][23][24][25] . The stages are defined qualitatively and do not have discrete boundaries, so interpreting smears is an inherently subjective art that requires training and experience [15][16][17][26][27][28] . The rat estrous cycle typically lasts four days, but the four stages vary in length from less than twelve hours to more than two days.…”

Section: Introductionmentioning

confidence: 99%

“…It is standard to exclude subjects with irregular cycles from experiments, but there is no standard definition of regular cycling. Exclusion criteria are therefore subjective, and perceptions that the cycle is highly sensitive to environmental conditions 16 and that irregularity is common 15 discourage estrous cycle tracking altogether. Semi-quantitative approaches [29][30][31] and machine learning tools 32,33 can reduce subjectivity in cytology interpretation, but a rigorous reporting framework and simpler methods that are accessible to the uninitiated are still needed.…”

Section: Introductionmentioning

confidence: 99%

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Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research

Raimondi

Eng

Kenny

et al. 2022

Preprint

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Despite known sex differences in brain function and incidence of neurological disorders, female subjects are routinely excluded from preclinical neuroscience research, particularly behavioral studies in rats. A common rationale for excluding females is that the hormone fluctuations of the estrous cycle will increase variability in experimental data. Accounting for the estrous cycle as an experimental variable requires expert knowledge of cycle tracking methods, which presents a barrier to widespread inclusion of female subjects. Conventional tracking relies on qualitative interpretation of vaginal cytology smears, and the subjective nature of this approach combined with a lack of reporting standards likely underlies the conflicting literature on estrous cycle effects on behavior. The estrous cycle is traditionally divided into stages based on cytology, but most stages do not directly reflect hormonal events and are therefore of limited relevance to neuroscience experiments. Here we present a simple, streamlined approach to estrous cycle monitoring in rats that eliminates subjective staging. Our method instead indexes the days of the estrous cycle to the one event that is unambiguously reflected in vaginal cytology: the pre-ovulatory surge in 17β-estradiol and subsequent epithelial cornification. With this tracking method, we demonstrate that cycle length is robustly regular across conditions. We quantified long-term memory in a Pavlovian fear conditioning experiment and uterine histology in a large cohort of rats, and found that grouping subjects by day was more sensitive in detecting cycle effects than grouping by traditional cytology staging. We present several datasets demonstrating the logic and applicability of our method, and show that, in the Track-by-Day framework, the cycle is highly regular and predictable in the vast majority of rats across a range of experimental conditions.

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Section: Discussioncontrasting

confidence: 78%

Section: Discussionmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research

Raimondi

Eng

Kenny

et al. 2022

Preprint

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The Track‐by‐Day Method for Monitoring the Rodent Estrous Cycle

2023

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The exclusion of female subjects from preclinical neuroscience research has traditionally been justified in part by concerns about potential effects of cycling ovarian hormones on brain function. There is evidence that some behavioral and neurobiological measures do change over the estrous cycle and, as the use of female subjects becomes increasingly routine, there is a greater demand for accessible cycle‐tracking methods. Conventional estrous cycle staging requires expert training in the qualitative interpretation of vaginal cytology smears, which serves as a barrier for novice researchers. In addition, definitions and reporting practices are not standardized across laboratories, which makes it difficult to compare results across studies and likely contributes to a false perception of the cycle as ephemeral and inconsistent. Here, we describe a streamlined method for monitoring the estrous cycle in rats, which we term Track‐by‐Day. It is simple to implement and inherently produces consistent reporting. Our protocol should serve to demystify and facilitate adoption of cycle tracking for those new to the practice. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Collection and staining of vaginal smears Basic Protocol 2: Track‐by‐Day classification of vaginal smears Support Protocol: Preparation of gelatin‐subbed slides

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Chronological and reproductive aging-associated changes in resistance to oxidative stress in post-reproductive female mice

et al. 2023

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

Cited by 5 publications

References 55 publications

Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research

Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research

The Track‐by‐Day Method for Monitoring the Rodent Estrous Cycle

Chronological and reproductive aging-associated changes in resistance to oxidative stress in post-reproductive female mice

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