How can the sex of mice be distinguished? - briefly
Males can be identified by the presence of palpable testes and a noticeably greater anogenital distance, whereas females lack testes and have a shorter distance. Additional confirmation is possible by genetic testing for the Y‑linked Sry gene.
How can the sex of mice be distinguished? - in detail
Distinguishing male from female mice relies on anatomical, physiological, and molecular criteria that become reliable at specific developmental stages.
In newborns, the most immediate indicator is the anogenital distance (AGD). Measure the space between the anus and the genital papilla; males exhibit a distance roughly twice that of females. Palpation of the genitalia reveals a small, recessed opening in females, whereas males possess a protruding preputial gland and a visible penile structure. These external features are discernible without magnification but require careful handling to avoid injury.
From weaning onward, secondary sexual characteristics emerge. Male mice develop larger, more prominent scent glands (e.g., the preputial and flank glands) and display a thicker, more muscular tail base. Females retain a smooth perineal region and lack pronounced glandular tissue. Coat coloration may differ in specific strains, but reliance on fur patterns alone is unreliable.
Behavioral observations provide supplementary clues. Males typically exhibit territorial aggression, mounting attempts, and urine marking. Females show estrous cycles, indicated by rhythmic changes in vaginal cytology. Vaginal smears taken with a sterile swab and examined under a microscope reveal epithelial cell types that correlate with cycle phases, confirming female status.
When visual assessment is insufficient—such as in genetically modified lines with ambiguous phenotypes—molecular methods are preferred. Polymerase chain reaction (PCR) targeting the Sry gene on the Y chromosome yields a binary result: presence confirms a male, absence indicates a female. DNA extraction from ear punches or tail clips supplies material for rapid amplification. Quantitative PCR can also detect copy-number variations in sex-linked genes for detailed genotyping.
Hormone assays offer an alternative biochemical approach. Serum testosterone concentrations rise sharply in males after puberty, while estradiol levels dominate in females. Enzyme-linked immunosorbent assays (ELISA) quantify these steroids with high sensitivity, though they require blood collection and proper timing relative to the estrous cycle.
For large colonies, automated systems integrate infrared imaging and machine learning to classify sex based on subtle morphological cues captured during routine handling. Such platforms reduce human error and increase throughput, but initial calibration against known-sex individuals remains essential.
Key procedural points:
- Perform AGD measurement within 24 hours of birth for maximal accuracy.
- Use gentle palpation to identify preputial glands in juveniles.
- Conduct vaginal cytology at consistent times to track estrous phases.
- Apply PCR for Sry detection when phenotypic markers are ambiguous.
- Verify hormone assay results with appropriate controls and sampling schedules.
Combining external examination, behavioral monitoring, and molecular diagnostics ensures reliable sex identification across developmental stages and experimental contexts.