Mice Gender: Anatomy, Genetics, and Behaviors

Sex differences in mice influence their anatomy, genetics, and behavior, making them a key consideration for pet owners, breeders, and researchers. Recognizing these differences is essential for proper care and for understanding how sex-linked traits affect biological and medical studies.

Male and female mice can be distinguished through physical characteristics, genetic testing, and behavioral observations. Each method offers valuable insights, particularly when considering strain-specific variations.

Visible Anatomical Differences

Male and female mice exhibit distinct physical traits that become more pronounced with maturity. One of the most reliable indicators is the anogenital distance—the space between the anus and genital opening. In males, this distance is about twice as long as in females, making it a widely used marker in laboratories and breeding programs, even in newborn pups.

As mice develop, additional features differentiate the sexes. Males possess external testes, which typically descend by 10 to 12 days after birth, housed within the scrotum and becoming more prominent with maturity. Females lack external reproductive organs, with their genital area positioned closer to the anus. The absence of a scrotal sac in females remains a consistent characteristic across strains.

Fur patterns and body size may provide clues but are less reliable than genital examination. Males in many strains tend to be slightly larger, with a broader head and more muscular build. However, diet, genetics, and environment can influence growth, making size an inconsistent determinant.

Nipple visibility is another distinguishing feature, particularly in young females. Around the first week of life, female pups exhibit small, evenly spaced nipples along the ventral side, which remain visible into adulthood. Males lack external nipples due to the suppression of mammary gland development during embryogenesis, making this absence a useful secondary marker when anogenital distance is unclear.

Noninvasive Genetic Testing Options

Advances in molecular biology have made genetic sex determination possible without invasive procedures. Noninvasive methods allow researchers and breeders to confirm sex using minimally collected samples like saliva swabs, fecal matter, or shed hair, preserving animal welfare while ensuring accuracy.

Polymerase chain reaction (PCR) is widely used for genetic sexing, amplifying DNA sequences linked to the Y chromosome, such as the Sry gene, which determines maleness. Extracting DNA from a noninvasive sample enables researchers to detect Y-linked markers, providing over 99% accuracy.

Droplet digital PCR (ddPCR) improves sensitivity by partitioning DNA into thousands of reactions, reducing false negatives and contamination, especially in low-concentration samples. A study published in Scientific Reports confirmed ddPCR’s near-perfect accuracy in identifying mouse sex from buccal swabs.

Next-generation sequencing (NGS) offers a more comprehensive approach by identifying chromosomal variations between sexes. While highly accurate, NGS is resource-intensive and typically reserved for large-scale genomic studies. A comparative analysis in PLOS ONE found that while NGS provides deeper genetic insights, PCR remains the preferred method for rapid and cost-effective sex identification in research and breeding.

Behavioral Cues in Juveniles and Adults

Sex-specific behaviors emerge early and become more pronounced with maturity. Juvenile males engage in frequent rough-and-tumble play, characterized by pouncing, pinning, and wrestling, influenced by early androgen exposure. Female juveniles, while still playful, tend to engage in more reciprocal grooming and huddling, reinforcing social bonds.

In adulthood, behavioral differences become more distinct, particularly in territoriality and mating interactions. Males establish dominance hierarchies through scent marking, driven by testosterone. Their urine contains pheromones that signal rank and deter rivals. Studies show male scent marks contain major urinary proteins (MUPs) that also attract females. Females use scent marking more selectively, often in response to reproductive status.

Vocalization patterns also differ, especially in courtship and social communication. Males emit ultrasonic vocalizations (USVs) when encountering females, producing complex, song-like calls that vary in pitch and duration. Research using spectrographic analysis shows males adjust their calls based on female responses. Females vocalize less frequently, mainly in maternal contexts, such as calling to pups.

Variations Across Mouse Strains

Sex-based differences in behavior and physiology vary by strain, as genetics shape traits like aggression, mating behaviors, and activity levels. These variations are particularly relevant in research, where strain-dependent differences can impact experimental outcomes.

C57BL/6 mice, widely used in biomedical research, show moderate sex differences in social and exploratory behaviors. Males are more territorial and engage in frequent scent marking, while females display stronger affiliative behaviors, such as communal nesting.

BALB/c mice, known for heightened anxiety-like behaviors, exhibit less pronounced sex-based differences in aggression but show variation in stress responses, with females generally more sensitive to environmental changes.

FVB/N mice display exaggerated sexual dimorphism in activity and cognitive performance. Males are significantly more active in open-field tests, while females outperform them in spatial learning tasks, suggesting differences in hippocampal function. These findings highlight how strain-specific genetics interact with sex to shape behaviors, making careful strain selection essential in experimental design.