Female mice exhibit less overall variance, with a higher proportion of structured variance, than males at multiple timescales of continuous body temperature and locomotive activity records

Abstract

Despite recent work demonstrating that female rodents and humans do not show greater variance in behavior and physiology than males due to ovulatory cycles, many researchers still default to using males in their investigations. Although government funding agencies now require inclusion of female subjects where applicable, the erroneous belief that the study of males reduces overall data variance continues to result in male subject bias. Recently, we reported the first direct experimental refutation of this belief by examining continuous body temperature and locomotor activity in male and female mice. These findings revealed that males exceeded female variance within and across individuals over time, showing greater variance within a day than females do across an entire estrous cycle. However, the possibility remains that male variance within a day is impacted by ultradian rhythms, analogous to the influence of infradian estrous cycles on female variance, and both sexes show predictable, structured variance across the day. If structures underlying variance can be predicted, then the variance can be statistically accounted for, reducing experimental error and increasing precision of measurements. Here we assess these continuous body temperature and activity data for the contributions of structured and unstructured variance to overall variance within and across individuals at ultradian, circadian, and infradian timescales. In no instance do females exceed male variance, and in most instances male variance exceeds female variance. Additionally, more female variance is accounted for by temporal structure. In conclusion, even when estrous cycles are not controlled for, females show less variability than males, and this advantage can be further capitalized upon by inclusion of known temporal patterns to control for previously unknown but structured sources of variance.

Why the estrous cycle matters for neuroscience

“…In sum, our data are consistent with the data previously reported in mice and rats that females are, on average, not more variable than males [21,22,26]. However, our data also clearly show that this finding is not, at all, predictive of whether the estrous cycle plays an important role in regulating the outcome of interest.…”

“…In fact, this variable was the major reason why females were excluded from neuroscience studies for decades [19]. In response, there was a concerted effort within the community to show that the variability of females is not higher than that of males [21,22,26] so that more people feel comfortable including females, avoiding expensive studies that may incorporate multiple female groups to account for varying sex hormones. So, would calls for incorporating the estrous cycle now bring us backwards?…”

“…This led to a trend that some researchers called "liberation of female animals" [22], implying that because females are similar to males in terms of variability, the estrous cycle can be dismissed, and that therefore females "deserve to be studied". And, this perceived need to justify the inclusion of females by rejecting female complexity, rather than by acknowledging a simple need for inclusivity, is still promoted to this day [23][24][25][26].…”

Cancer treatment induces neuroinflammation and behavioral deficits in mice

“…Female SKH1 mice were evaluated in this study as CRCI is overrepresented in female breast cancer patients, and because women have been shown to have greater cognitive impairment than men following surgery for early-stage colorectal carcinoma (Vardy et al, 2014 ; Santos and Pyter, 2018 ). Moreover, female mice have been shown to have less unstructured variance in temperature and activity compared to males, reducing potential uncertainty in behavior during testing within the study groups (Smarr and Kriegsfeld, 2022 ). Non-tumor bearing mice were selected to isolate treatment-related changes from tumor changes as cancer is known to cause cognitive changes (Janelsins et al, 2011 ; Santos and Pyter, 2018 ; Lange et al, 2019 ).…”

Battle of the sexes: who is more variable, and does it really matter?

“…Because years after the introduction of policies to equalize female and male representation in animal studies 2 , studies in males still outnumber those in females 3 . Moreover, despite multiple meta-analyses 3 – 6 and individual studies 7 demonstrating that data from females are not, as a rule, more variable than male counterparts, scientists in this decade are still stating that females are more variable than males as their major justification for all-male studies 3 . The Levy et al 1 study is another nail in the coffin of the “greater female variability” trope; and a mighty nail at that, because motion sequencing enabled nuanced, dynamic aspects of behaviour to be captured, affording high sensitivity.…”