How to Distinguish a Male Rat from a Female

Introduction to Rat Sexing

Why Sexing is Important

For Pet Owners

Pet owners who need to determine the sex of a domestic rat should focus on three reliable indicators: genital anatomy, nipple development, and size differences.

The most definitive characteristic is the shape of the genital opening. In males, the distance between the anus and the genital papilla (the “anogenital distance”) is noticeably larger, often twice the length seen in females. A small, round protrusion, the testicular sac, may be visible under the tail in mature males. Females display a vertical slit directly behind the anus, without a pronounced gap.

Nipple visibility offers a secondary clue. Female rats develop a line of small nipples along the ventral surface, usually apparent after four weeks of age. Males either lack nipples or have them extremely reduced, making them difficult to detect without magnification.

Body size provides a tertiary reference. Adult males tend to be slightly heavier and longer than females of the same age, though overlap is common and this trait should not be used in isolation.

Practical checklist for sex identification

Measure the anogenital distance with a ruler; a gap greater than 1 cm suggests a male.
Observe the shape of the genital opening: round papilla for males, vertical slit for females.
Examine the underside for a row of nipples; presence indicates a female.
Look for testicular sacs under the tail in mature rats; visible sacs confirm a male.

When handling a rat, use gentle restraint to prevent stress. Conduct the examination in a well‑lit area, and consider using a magnifying glass for small juveniles. Accurate sex determination aids in proper breeding management, health monitoring, and behavioral expectations for pet owners.

For Breeders

Breeders must reliably determine the sex of young rats to maintain accurate breeding records and avoid unwanted litters. Early identification relies on observable anatomical features that become distinct within the first two weeks of life.

Anogenital distance: Males exhibit a noticeably wider gap between the anus and the genital opening compared to females. Measure with a fine ruler or by visual estimation; a distance greater than 5 mm typically indicates a male.
Presence of testes: In pups older than 10 days, small, creamy‑white bulges appear in the scrotal area. Gently press the abdomen; testes will descend into the scrotum in males, while females retain a smooth abdomen.
Nipple development: Female pups develop prominent nipples by day 12. Males possess faint, flat nipples that are difficult to discern without magnification.

Sex differentiation becomes more reliable after weaning (21 days). Behavioral cues can supplement physical examination but should not replace it.

Practical steps for a breeding operation:

Separate litters by age, examining each pup under a bright, magnified light source.
Record the measured anogenital distance for every individual in a logbook.
Mark identified males with a small, non‑invasive tattoo or ear tag; females receive a different color tag.
Re‑examine all animals at three weeks to confirm initial assessment, correcting any discrepancies before pairing.

Accurate sexing prevents accidental breeding and supports selective line development. Maintaining detailed records of each identification event ensures traceability throughout the breeding cycle.

For Scientific Research

Sex identification in laboratory rats is a prerequisite for experimental design, data interpretation, and compliance with ethical standards. Accurate determination of male or female status eliminates confounding variables and ensures reproducibility across studies.

Morphological assessment provides the quickest initial indication. Key external features include:

Anogenital distance (AGD): males exhibit a greater AGD measured from the anus to the genital papilla.
Genital morphology: the presence of a visible scrotum and testes in males; females display a vaginal opening with a less pronounced papilla.
Nipple pattern: males typically lack prominent mammary buds, whereas females possess a full complement of nipples along the ventral line.

Timing of observation influences reliability. Recommended stages are:

Neonatal (postnatal day 1–3): AGD measurement yields the highest sex discrimination accuracy.
Weaning (postnatal day 21): genital structures become more distinct, facilitating visual confirmation.
Adult (postnatal day 60+): full development of secondary sexual characteristics allows definitive classification.

Molecular techniques complement visual methods and resolve ambiguous cases. Established protocols include:

Polymerase chain reaction targeting the Sry gene, which is exclusive to the Y chromosome.
Fluorescence in situ hybridization (FISH) for chromosome-specific probes.
Quantitative PCR for sex‑specific gene expression patterns.

Best practice integrates both phenotypic and genotypic data. Initial morphological screening narrows the cohort, while molecular verification confirms sex with near‑absolute certainty. This dual‑approach minimizes misclassification risk and supports rigorous scientific inquiry.

Primary Physical Indicators

Anogenital Distance

Visual Inspection

Visual inspection provides the quickest means of determining a rat’s sex. The examiner evaluates external characteristics that differ between males and females.

Key external features:

Anogenital distance – the space between the anus and the genital opening is noticeably longer in males. In females the distance is short, with the genital papilla situated close to the anus.
Genital morphology – male rats display a visible scrotum containing two testes, often appearing as a small, rounded bulge. Female rats lack a scrotum; instead, a single, shallow vulvar opening is present, sometimes accompanied by a thin, hair‑covered labia.
Nipple development – adult females develop prominent nipples, especially on the ventral surface. Males retain only faint, undeveloped nipples that are difficult to see without magnification.

Procedure for accurate visual sexing:

Secure the rat gently but firmly, exposing the lower abdomen and hindquarters.
Observe the anogenital region from a side view to measure the distance between the anus and genital opening.
Identify the presence or absence of a scrotal sac; if present, confirm the visibility of testes.
Check for nipple prominence if the animal is mature; this supports female identification.

Age influences reliability. Neonates exhibit minimal genital differentiation; the anogenital distance remains the most dependable indicator until the testes descend, typically by 10‑14 days of age. After weaning, all listed characteristics become clearly distinguishable.

Measurement Techniques

Accurate sex determination in laboratory rats relies on precise measurement methods that minimize ambiguity and support reproducible results.

Physical measurements provide the quickest assessment. The anogenital distance (AGD)—the space between the anus and the genital papilla—differs markedly between sexes; males exhibit a longer AGD. Use calibrated digital calipers to record AGD to the nearest 0.01 mm, and compare values against established reference ranges for the specific strain and age.

External genital examination complements AGD data. In males, the preputial fold encloses a visible penis, while females display a simple vaginal opening without a preputial sheath. Employ a magnifying lens (≥10×) and a fine-tipped probe to confirm anatomical features without causing tissue damage.

Body weight and growth curves serve as secondary indicators. Male juveniles typically gain weight faster after weaning. Record daily weight on an analytical balance with 0.01 g resolution, then plot against age‑matched growth standards to identify sex‑related deviations.

Tail length measurement offers an additional metric; males often possess slightly longer tails relative to overall body length. Measure tail from the base to the tip using a flexible measuring tape, then calculate the tail‑to‑body ratio for comparison with sex‑specific benchmarks.

Molecular techniques provide definitive confirmation when phenotypic measurements are inconclusive. Extract genomic DNA from a small ear punch and perform polymerase chain reaction (PCR) targeting the Sry gene, the male‑specific sex‑determining region on the Y chromosome. Gel electrophoresis visualizes the presence or absence of the Sry amplicon, delivering unequivocal sex identification.

Summary of recommended measurement techniques

Anogenital distance: digital calipers, 0.01 mm precision.
External genitalia: magnification, preputial fold observation.
Body weight: analytical balance, 0.01 g precision, growth curve analysis.
Tail length: flexible tape, tail‑to‑body ratio calculation.
Genetic testing: DNA extraction, Sry‑specific PCR, electrophoretic detection.

Applying these methods in combination ensures reliable sex discrimination, supports experimental integrity, and aligns with best practices for rodent research.

Genital Anatomy

Male Characteristics

Male rats display several distinct physical and behavioral traits that separate them from females.

Externally, the most reliable indicator is the presence of testes, visible as a small, oval-shaped bulge beneath the tail. This structure becomes apparent once the animal reaches sexual maturity, typically around six weeks of age. The anogenital distance—the space between the anus and the genital opening—is noticeably greater in males than in females. In mature specimens, the distance measures roughly twice that of a female of comparable size.

The fur pattern can also provide clues. Males often exhibit a more pronounced dorsal stripe, especially in strains where this trait is genetically linked to the Y chromosome. In addition, male rats tend to have larger, broader heads and a more robust body mass relative to females of the same age.

Behaviorally, males are more likely to display territorial marking. Urine spraying and frequent scent‑marking with glandular secretions occur regularly, especially when a male encounters unfamiliar conspecifics. Aggressive interactions, such as mounting and wrestling, appear more frequently among males, particularly during the breeding season.

A concise checklist of male characteristics:

Visible testes beneath the tail after sexual maturity
Longer anogenital distance (approximately double that of females)
Broader head and increased overall body mass
Pronounced dorsal stripe in certain genetic lines
Frequent urine spraying and scent marking
Higher incidence of aggressive and mounting behaviors

These attributes, taken together, provide a reliable framework for identifying male rats without reference to the overarching identification method.

Testicles

Testicles are the primary external indicator of a male rat’s sex. In mature individuals they appear as paired, oval structures situated within the scrotal sac, directly beneath the base of the tail. The scrotum is typically pink‑to‑light‑brown, thin‑walled, and may be visible through the animal’s fur when the coat is not overly dense.

Key characteristics of rat testicles:

Location – positioned posterior to the ventral abdomen, on either side of the anus.
Size – each testis measures roughly 5–7 mm in length in adult males; they are proportionally larger than the surrounding tissue.
Texture – smooth, firm to the touch, and separated by a thin membrane.
Visibility – the scrotal sac becomes more prominent as the rat reaches sexual maturity, usually after 5–6 weeks of age.

In contrast, females lack any scrotal sac and present a smooth, uninterrupted ventral surface. Absence of testicular bulges, combined with the presence of a genital papilla rather than a scrotum, confirms the animal is female.

Penis Sheath

The penis sheath, also called the preputial pouch, is the most reliable external indicator of a male rat. It appears as a thin, translucent fold of skin covering the tip of the genitalia. In a mature male, the sheath can be gently lifted to reveal a small, cylindrical organ approximately 3–5 mm in length. The organ is firm, slightly curved, and may be visible as a faintly darker structure within the sheath. Female rats lack this pouch entirely; the ventral surface of the perineal region is smooth, without any protruding skin fold or underlying organ.

When examining a rat:

Gently press the lower abdomen to expose the perineal area.
Look for a recessed, tube‑like opening at the base of the sheath; its presence confirms maleness.
In the absence of the sheath, the area will consist of a simple fur‑covered skin surface, indicating a female.

The penis sheath develops fully by four weeks of age; younger pups may not yet display a distinct pouch, making sex identification more challenging. In such cases, reliance on other characteristics—such as anogenital distance—becomes necessary, but the sheath remains the definitive marker once it is present.

Female Characteristics

Female rats display a set of anatomical markers that reliably separate them from males. The most definitive indicator is the presence of a genital opening (vulva) located ventrally, just behind the anal slit, whereas males possess a scrotum and a markedly larger anogenital distance. Additional characteristics include:

Prominent nipples along the ventral surface, evident from weaning onward; males lack functional mammary tissue.
Smaller overall body mass and slightly shorter tail relative to body length, especially in mature individuals.
Absence of a scrotal sac; the abdomen remains smooth without the bulge typical of male testes.
Distinct pheromonal profile produced by the estrous cycle, detectable by other rodents but not by human scent.
Behavioral cues such as nesting activity and reduced aggressive displays, though these can vary with environment.

These traits, examined together, allow precise identification of female rats in laboratory, veterinary, or pest‑management contexts.

Vaginal Opening

The vaginal opening is the primary external indicator of a female rat. It appears as a small, circular slit located just posterior to the anus, typically 2–3 mm in diameter in adult specimens. The surrounding tissue is smooth and lacks the prominent scrotal sac or testicular bulge seen in males. In juvenile rats the opening may be less distinct, but it remains visible as a faint line or shallow groove, whereas males display a single, central urethral opening without surrounding labial folds.

When assessing sex, gently restrain the animal and view the ventral side with adequate lighting. Identify the following features:

Presence of a circular or oval opening flanked by labia; absence of scrotal tissue.
Lack of palpable testes within the scrotal area.
Position of the opening relative to the anus: females have the opening immediately behind the anal vent, while males exhibit a single urethral opening directly adjacent to the anus.

Potential sources of error include swollen or injured perineal tissue, which can obscure the opening, and immature females whose genitalia are not fully developed. In such cases, supplementary characteristics—such as the shape of the preputial gland or the presence of a penis—should be examined.

Accurate identification of the vaginal opening enables reliable sex determination, essential for breeding programs, research protocols, and health assessments.

Urethral Opening

The urethral opening provides a reliable visual cue for sex identification in laboratory and pet rats. In males, the opening is a distinct, circular slit located on the underside of the prepuce, visible as a small, darkened aperture that aligns with the tip of the penis. In females, the urethral opening is positioned near the ventral surface of the abdomen, close to the genital papilla, and appears as a tiny, inconspicuous pore without surrounding penile tissue.

Key observable differences:

Position: male – at the distal end of the prepuce; female – near the ventral abdominal wall.
Shape: male – circular slit; female – minute pore.
Surrounding structures: male – accompanied by the penile shaft and scrotal folds; female – adjacent to the vulvar region without penile anatomy.

Accurate assessment of these characteristics enables rapid, non‑invasive determination of rat sex, supporting breeding programs, experimental design, and health monitoring.

Secondary Physical Indicators

Body Size and Build

Typical Male Proportions

Male rats exhibit a set of proportional traits that differ consistently from those of females. These traits become more evident after the animal reaches sexual maturity.

Body length typically exceeds tail length by 10‑15 %, whereas females often have a tail equal to or longer than the body.
Head width measures roughly 20‑25 % greater than that of a comparable female, giving the skull a broader appearance.
Shoulder width is noticeably broader, creating a more rectangular torso silhouette.
The distance between the anal opening and the genital papilla is greater, reflecting a longer ventral cavity.

These measurements provide a practical framework for sex identification. Observing several of the listed proportions together reduces the likelihood of misclassification.

Typical Female Proportions

Female rats display a set of proportional traits that differ consistently from those of males. These traits are most reliable when the animal is fully grown and not pregnant.

Body length (nose‑to‑base of tail): 18–22 cm, slightly shorter than the average male.
Weight: 150–250 g, lower than the typical male range of 200–300 g.
Tail length: 10–12 cm, representing about 55 % of total body length; males usually exceed 60 %.
Head width: narrower, with the distance between the eyes averaging 1.0 cm versus 1.2 cm in males.
Pelvic width: broader relative to overall body size, measured at 1.5 cm across the hips compared with 1.3 cm in males.
Hind‑foot size: 1.8 cm in length, marginally smaller than the male average of 2.0 cm.

These dimensions hold true across common laboratory strains such as Sprague‑Dawley and Wistar. Juvenile rats have overlapping measurements, so proportion‑based identification should be confined to adults. Pregnancy temporarily alters abdominal girth, but the underlying skeletal proportions remain unchanged, preserving the diagnostic value of the listed metrics.

Nipple Presence

Female Nipple Visibility

Female rats develop a line of eight to twelve mammary buds along the ventral abdomen. In mature females, each bud enlarges into a nipple that protrudes slightly above the surrounding fur. The nipples are most evident between the forelimbs and the hindlimbs, forming a symmetrical row. Visibility increases after weaning, peaks during estrus, and becomes most pronounced when the animal is pregnant or lactating, as the glands swell and the skin stretches.

Key characteristics of female nipple visibility:

Location: Mid‑ventral line, roughly midway between the ribcage and the pelvis.
Number: Typically eight to twelve, arranged in a single row.
Shape: Small, rounded protrusions with a darker pink or reddish hue compared to surrounding fur.
Size variation: Enlarged during pregnancy and nursing; reduced but still detectable in non‑reproductive adults.
Contrast: More noticeable on light‑colored fur; may require gentle illumination on dark‑fur specimens.

Male rats possess the same embryonic mammary buds, but these remain undeveloped. In adult males, the ventral abdomen shows a smooth, fur‑covered surface without discernible protrusions. Any faint markings are usually hidden beneath the coat and lack the pink coloration typical of functional nipples.

When assessing sex, examine the ventral abdomen for the presence, number, and coloration of nipples. Confirm findings with additional traits—such as genital morphology or body size—to reduce misidentification, especially in juvenile or lightly furred individuals.

Male Nipple Absence

Male rats do not develop functional nipples, whereas females possess a complete set of eight. The absence is evident after weaning, when the mammary ridge in males regresses and the skin remains smooth. To use this trait for sex identification, follow these steps:

Gently lift the fur along the ventral midline, starting just behind the forepaws and extending to the abdomen.
Observe the skin texture; a smooth surface without raised papillae indicates a male.
Confirm the presence of nipples in the same region; visible nipples confirm a female.

The lack of nipples results from androgen‑driven suppression of mammary tissue during embryogenesis. This morphological difference is consistent across common laboratory strains and wild populations, providing a reliable, non‑invasive marker for distinguishing sexes in adult and juvenile rats.

Behavioral Cues

Social Interactions

Dominance Displays

Dominance displays are a reliable behavioral marker when separating male and female rats. Males typically establish hierarchy through overt actions, while females exhibit subtler patterns.

Territorial scent marking – Males frequently deposit urine on the cage floor and objects, creating conspicuous chemical trails. Females produce fewer marks and concentrate them near nesting sites.
Chasing and mounting – Adult males regularly initiate pursuits and mount conspecifics as a claim to status. Female rats may display brief mounting during estrus but lack persistent aggressive chasing.
Boxing and wrestling – Male rats engage in prolonged bouts involving forelimb strikes and body slams. Female encounters are shorter, often limited to brief pushes without sustained aggression.
Vocalizations – High‑frequency squeals accompany male confrontations; female vocal output remains low and infrequent during social disputes.

Observation of these behaviors over several days yields a clear sex distinction, especially when combined with physical cues such as genital morphology. Consistent dominance expression strongly suggests a male rat, whereas its absence or minimal presence points to a female.

Mating Behaviors

Mating conduct offers reliable cues for determining the sex of a rat. Observers can separate males and females by noting distinct actions that emerge during the reproductive period.

Male rats exhibit the following behaviors:

Frequent urine marking of objects and cage walls.
Persistent attempts to mount any conspecific, regardless of the partner’s sex.
Aggressive patrol of the enclosure, often accompanied by dorsal grooming.
Production of high‑frequency ultrasonic calls during courting bouts.

Female rats display a contrasting set of actions:

Adoption of the lordosis posture when approached by a male, characterized by a raised rear and lowered abdomen.
Receptive vocalizations at a lower ultrasonic frequency than those of males.
Construction of nest material and increased grooming of the ventral area during estrus.
Reduced territorial marking; focus shifts to social interaction with the courting male.

When monitoring a group, note the timing of estrous cycles: females become receptive for a brief window each 4–5 days, during which the listed female behaviors intensify. Males maintain courting and marking activities throughout the cycle, providing a continuous behavioral signature.

By systematically recording these patterns, sex identification can be achieved without reliance on physical examination alone.

Scent Marking

Male Scent Glands

Male rats develop a pair of scent glands, known as the preputial and flank glands, that are absent or rudimentary in females. The preputial gland resides near the base of the penis, producing a waxy secretion that coats the fur and leaves a faint musky odor. The flank gland appears as a small, raised patch of skin along the lateral abdomen, often darkened by pigment and secreting a distinct, pungent scent.

These glands serve as reliable external markers for sex identification. Key characteristics include:

Presence of a visible, slightly raised flank gland on each side of the torso.
Darkened or glossy skin over the flank gland area.
Detectable musky odor when the animal is handled or when the cage is inspected.
Absence of a preputial gland in females; any similar structure near the genital region indicates a male.

During routine observation, examine the lateral abdomen for the flank gland and note any associated odor. In laboratory settings, a quick swab of the flank area can confirm the presence of male-specific secretions, providing definitive sex determination without invasive procedures.

Female Scent Glands

Female rats possess a pair of specialized scent glands located near the vulva, commonly referred to as the vaginal or perineal glands. These glands secrete a milky, slightly oily fluid that becomes most pronounced during estrus. The secretion contains pheromones that attract males and signal reproductive readiness. Anatomically, the glands are small, translucent sacs embedded in the subcutaneous tissue and are visible as a subtle swelling on the ventral side of the abdomen, just anterior to the anus.

When comparing to male counterparts, the following characteristics are reliable for sex identification:

Absence of a prominent preputial gland, which in males appears as a larger, lobulated structure near the penis.
Presence of a ventral swelling that enlarges cyclically in females, especially during the fertile phase.
Detection of a distinct, sweet‑to‑musky scent emanating from the female’s perineal region, absent in males.
Lack of the pronounced, musky odor produced by the male’s scrotal and preputial secretions.

Observing these glandular differences, alongside other morphological cues, enables accurate determination of a rat’s sex without invasive procedures.

When to Sex a Rat

Optimal Age for Identification

Neonatal Stage

The neonatal period provides the earliest reliable indicators for sex determination in laboratory rats. At birth, anatomical differences are measurable without invasive procedures.

Anogenital distance (AGD) is markedly longer in males than in females. Calibrate a digital caliper to the ventral surface of the abdomen, then measure from the anus to the genital papilla; values exceeding the species‑specific threshold indicate a male.
Testes are visible in male pups as paired, creamy‑white masses beneath the abdominal wall. In females, the corresponding region lacks palpable tissue and appears smooth.
Nipples are present in both sexes, but males retain a full complement of twelve, while females display only six. Count the nipples along the ventral line to corroborate the sex assignment.

These characteristics remain consistent through the first week of life, allowing accurate classification before sexual maturity.

Juvenile Stage

During the juvenile period (approximately 3‑5 weeks of age) external sexual characteristics are not fully developed, yet reliable indicators allow sex determination.

The anogenital distance (AGD) measures the space between the anus and the genital opening. In males the AGD is roughly twice that of females. Use a fine ruler or calipers to compare the distance to established reference values for the specific strain.
Pre‑putial folds appear in males as a small, raised ridge surrounding the genital papilla. In females the area remains flat and smooth.
Testes are not yet descended but can be palpated as a firm, pea‑sized mass beneath the abdominal wall, just posterior to the ventral midline. Absence of such a mass indicates a female.
Nipples are present in both sexes, but the number and spacing differ slightly; however, this trait alone is unreliable for juveniles.

Accurate sexing requires gentle handling to avoid stress. Record measurements and observations promptly, as the juvenile window closes rapidly when secondary sexual traits become more pronounced.

Adult Stage

In adult rats, sexual dimorphism becomes reliably apparent through external anatomy, secondary characteristics, and behavioral cues.

The most direct indicator is the anogenital region. Males possess a visible scrotum containing testes; the scrotal sac lies ventral to the anus and is covered by a thin, pinkish skin. Females lack a scrotum; instead, a single, small opening (the vulva) appears just behind the anus, without any surrounding swelling. The distance between the anus and genital opening (anogenital distance) is markedly greater in males, often exceeding 1 cm, while females exhibit a distance of less than 0.5 cm.

Secondary traits develop after sexual maturity:

Nipples: Both sexes have nipples, but in adult males the nipples are reduced in size and may be recessed, whereas females retain more prominent, functional nipples.
Sebaceous (preputial) glands: Males develop larger, more active preputial glands that produce a musky odor used in territorial marking; females have smaller, less active glands.
Body mass: Adult males typically weigh 20–30 % more than females of the same strain, reflecting greater muscle development.
Tail length: Males often exhibit a slightly longer tail relative to body size.

Behavioral observations support anatomical assessment. Adult males display frequent mounting, aggression toward conspecifics, and territorial scent marking. Females exhibit nesting behavior, maternal care, and less overt aggression.

When confirming sex in the adult stage, combine visual inspection of the anogenital area with measurement of anogenital distance, assessment of secondary characteristics, and, if necessary, observation of sex‑specific behaviors. This multi‑factor approach yields accurate identification.

Considerations for Accuracy

Lighting Conditions

Accurate sex identification of laboratory rats depends on clear visual access to genital morphology, and lighting conditions directly affect that visibility. Bright, uniform illumination eliminates shadows that can obscure the anogenital region, allowing reliable observation of the preputial fold in males and the vaginal opening in females. Excessive glare or uneven light creates reflections on fur, reducing contrast and increasing the risk of misclassification.

Optimal lighting parameters include:

Light intensity of 500–800 lux measured at the observation plane; lower levels diminish detail, higher levels cause glare.
Color temperature between 4,000 K and 5,500 K, providing a neutral white spectrum that renders fur and skin tones without color distortion.
Diffused sources such as LED panels with matte diffusers to spread illumination evenly across the animal’s body.
Minimal background illumination to prevent competing light sources from generating shadows.

When using artificial light, verify that the spectrum does not contain strong ultraviolet peaks, which can cause fur fluorescence and mask anatomical features. Consistent positioning of the light source at a 45‑degree angle to the animal’s side offers the best view of the genital area while preserving depth perception. Adjusting these variables yields reproducible conditions for distinguishing male from female rats.

Handling Techniques

When examining a rat to determine sex, handling must be precise and humane. Proper technique reduces stress for the animal and improves the visibility of distinguishing features.

Begin by washing hands and wearing disposable nitrile gloves. Secure a clean work surface and keep a towel or soft pad ready for the animal. Approach the rat calmly; sudden movements can trigger a defensive response.

To restrain the animal, place it on its back using a gentle “hand‑over‑hand” method: one hand supports the chest while the other hand gently slides under the hindquarters. Hold the rat with the thumbs positioned on the sides of the neck and the fingers wrapped around the torso, forming a secure but non‑tight grip. Maintain this position for no more than a few seconds.

Once restrained, locate the anogenital region. The male’s genital papilla protrudes as a small, rounded bulge between the anus and the urinary opening, while the female presents a smooth, flat area with a visible opening. Use a magnifying lens if necessary to enhance detail.

After assessment, release the rat by allowing it to slide forward onto the pad, then gently lift it onto a clean cage or transport container. Observe the animal for a brief period to ensure normal movement before returning it to its enclosure.

Key handling points:

Clean hands and gloves protect both handler and rat.
Soft, stable surface prevents injury.
Brief, controlled restraint minimizes stress.
Direct visual inspection of the anogenital area provides reliable sex identification.
Immediate release restores normal behavior.

Consistent application of these practices yields accurate results while maintaining animal welfare.