Introduction

Guinea pigs (Cavia porcellus) have served as an important animal model in biomedical research since the early 19th century, contributing to numerous foundational discoveries and therapeutic advances. They are particularly valuable in areas where their physiological, immunological, or developmental features more closely resemble those of humans than those of other commonly used rodent models such as mice or rats  [1]. Notably, guinea pigs played a central role in pioneering studies on infectious diseases, including bacterial [2], pulmonary [3], and sexually transmitted infections [4]. Owing to their anatomical and neurodevelopmental similarities to humans, especially within the auditory system, guinea pigs have become an important model for auditory research  [5]. Their use by researchers such as Robert Koch [6] and Emil von Behring [7] contributed to landmark discoveries that were later recognized with Nobel Prizes. Beyond infectious disease and sensory research, guinea pigs are also increasingly recognized as a powerful model for studying prenatal and postnatal development [8,9]. Since developmental processes cannot be easily investigated in humans, animal models play a critical role in this field. Guinea pigs are particularly well suited for such studies due to key similarities in placental structure, implantation patterns, neuronal development, and pregnancy progression [10,11]. As a result, they have been widely used in studies investigating preeclampsia, placental barrier function, and placental transfer [12–15].

Although guinea pigs have been studied extensively in the context of developmental biology and reproduction, many procedures, such as standardised protocols for successful timed mating, pregnancy confirmation and embryonic development remain limited in the literature. Given the comparatively long estrous cycle (15–17 days) and gestational period in guinea pigs, as well as the narrow ovulation window of only a few hours, precise timing of mating is essential to achieve successful pregnancies [16]. While external signs of receptivity, such as vaginal membrane perforation, can be used to guide mating, these require regular and careful monitoring by trained personnel [17]. For developmental studies, timed mating is particularly critical to reliably stage embryos across gestation. However, existing literature largely describes prolonged cohabitation or mating windows over several days, which impairs precise staging. In this protocol, we describe an approach to increase timed mating efficiency in guinea pigs. Our method builds upon and extends established techniques by Wilson et al. (2021) and others, combining daily assessment of the vaginal membrane with vaginal cytology [16–19]. In contrast to mice, where mating can be confirmed by the presence of a vaginal plug, such identification in guinea pigs is unreliable: plugs may be expelled shortly after mating or not be detectable at all [16,17]. Thus, we provide a decision tree that can be used by unexperienced scientist to achieve efficient timed mating.

Furthermore, gestational weight gain becomes a reliable pregnancy indicator only in mid-gestation. While one study reports that pregnancy can be confirmed via ultrasound as early as embryonic day 16 (E16) [20], more recent studies describe reliable detection from approximately E20 onward [17,21]. To enable early treatment paradigms that can be tested in guinea pigs during the first trimester, we aimed to improve pregnancy detection using various ultrasound devices and successfully detected gestational sacs as early as E12. Additionally, we provide morphological documentation of embryonic development from E15 to E27.

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io, https://dx.doi.org/10.17504/protocols.io.kqdg31xpql25/v1 and is included for printing as supporting information file 1 with this article.

Expected results

Guinea pigs offer several advantages as animal models for developmental research; however, researchers unfamiliar with the species may struggle with key procedures for successful mating and pregnancy detection. Unlike mice, in which the estrous cycle can be assessed by simple visual inspection of the external genitalia [22], a widely accepted and practiced method, the same is not straightforward in guinea pigs. In guinea pigs, the vaginal closure membrane, composed of epithelial cells and sealing the vaginal orifice during non-fertile phases [16], serves as a useful marker of estrous stage. However, the membrane is not easily visible without gently parting the labia (Fig 1), a procedure requiring familiarity and training. For untrained personnel, the open phase, when the membrane is absent or perforated, is typically the easiest to recognize. Regular monitoring of the vaginal membrane can improve mating efficiency, yet mis-timed matings may still occur since ovulation can precede the membrane’s rupture. As originally described by Stockard and Papanicolaou, vaginal secretions, specifically their presence and color, can offer additional cues for optimal mating timing [16]. These secretions are also detectable as mucus films on vaginal cytology smears.

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Fig 1. Examination of the vaginal membrane.

https://doi.org/10.1371/journal.pone.0347318.g001

Vaginal cytology further supports accurate estrous cycle staging. As early as 1917–1919, Stockard and Papanicolaou described four distinct stages based on gradual changes in the vaginal epithelium, with ovulation occurring shortly before leukocyte infiltration of the vaginal wall [16,23]. Copulation is typically restricted to the phase when leukocytes are absent, which is further subdivided into a preparatory phase (when the vaginal lumen appears dry) and a subsequent phase marked by the onset of mucus secretion, corresponding to the „first half” and „second half” during the “closed” stage. Leukocyte migration through the uterine and vaginal epithelium begins in the second phase, corresponding to the “opening” stage, and leukocytes remain prominent during subsequent stages alongside epithelial cells. If fertilization does not occur, early detection of non-pregnancy is essential to make timely use of the subsequent ovulatory period. In mice, detection of a copulatory plug the morning after mating is standard; however, plug formation in guinea pigs remains debated. In our observations, a true copulatory plug was never identified, though seminal residue was occasionally visible on the vaginal opening. It remains unclear whether guinea pigs do not form plugs, whether plugs are expelled rapidly, or whether identification requires more advanced training.

As an alternative, we attempted to detect sperm via PCR targeting the Y-chromosome using previously published primers for Dystrophin and Sry [24]. These were validated on male and female liver tissue as well as sperm isolated from testicular tissue. Despite optimization via temperature gradient testing and extended cycling protocols, swabs from the vaginal opening post-mating failed to yield sufficient material for sperm detection, even in cases where mating was confirmed by resulting pregnancy. Therefore, the combination of visual inspection of the estrous cycle (Fig 2), vaginal cytology (Fig 3) and ultrasound remains the most reliable method for early pregnancy confirmation. While previous studies reported that ultrasound detection of pregnancy is possible by E16 [20], our findings indicate it is feasible as early as E12 (Fig 4). At this stage, while direct visualization of embryos or placental tissues may be challenging, gestational sacs can be clearly identified due to their thick-walled structure. Visualization of both placental tissue and the embryo is more challenging and may only be achieved by imaging from multiple angles at this early stage. Accurate identification requires scanning the correct anatomical region, as large blood vessels can resemble gestational sacs (S2 File).

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Fig 2. Changes in the vaginal membrane during different phases of the estrous cycle.

(A) Closed vaginal membrane: The female external genitalia form a visible trident shape when the vaginal membrane is closed. Opening: The vaginal membrane begins to separate, and the clitoris appears swollen and changes colour from pale to a darker pink. Open: The vaginal membrane is fully perforated and the external vaginal opening is visible as a dark pink opening. Closing: The vaginal membrane begins to close from the outer edges, and the swelling subsides. Filled arrowhead: urethral opening, open arrow: vaginal membrane, filled arrow: anus, open arrowhead: vaginal opening. (B) Duration of each phase of the estrous cycle based on changes in the vaginal membrane (each dot represents one animal; mean ± SD).

https://doi.org/10.1371/journal.pone.0347318.g002

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Fig 3. Vaginal cytology as a measure of fertility.

(A) Scoring system based on the relative presence of different vaginal cell types. (B) Decision-making scheme to assess the likelihood of a positive pregnancy outcome. (C) Representative vaginal smears from the day of mating in one pregnant and one non-pregnant animal. In the pregnant animal, fragmented cells and mucus are visible, whereas in the non-pregnant animal, large, well-defined cells predominate. (D) Distribution of cell types on the day of mating in animals that were later confirmed to be pregnant or failed to conceive. (E) Changes in cell type distribution one day after mating in animals that were later confirmed to be pregnant or failed to conceive.

https://doi.org/10.1371/journal.pone.0347318.g003

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Fig 4. Ultrasound examination of embryos at E12.

(A) Schematic showing the anatomical position of internal organs and the pregnant uterus. The dashed outline indicates the area that must be shaved prior to ultrasound examination. (B) Ultrasound images from two pregnant animals obtained using the VScan Air CL, the filled arrowhead indicates a visible embryo. (C) Ultrasound images from two pregnant animals obtained using the Butterfly iQ + Vet, the open arrow indicates a visible placenta.

https://doi.org/10.1371/journal.pone.0347318.g004

By approximately E20, visualization of the embryo and placenta becomes increasingly distinct, eliminating the need to rely solely on gestational sac visualization (S3 File). In later gestation (around E36), the embryo has developed into a fetus with distinguishable anatomical features. At this stage, ultrasound can detect the spine, other skeletal structures, and the fetal heartbeat (S4 File) [20]. During the second half of gestation, as maternal weight gain becomes more pronounced, pregnancy can also be confirmed by cautious abdominal palpation.

For developmental studies, embryos must be carefully dissected from the uterus by incising the uterine wall using blunt-tipped scissors to minimize tissue damage. Embryos are attached to the decidua via the subplacenta, from which they must be gently separated. Each embryo is enclosed within the parietal yolk sac (Fig 5A-D), through which it remains connected to the placenta via the umbilical cord. Isolation of the embryo requires the stepwise removal of multiple tissue layers. The outermost layer is the highly vascularized parietal yolk sac, followed by the visceral yolk sac. The embryo resides within the amniotic cavity, enclosed by the amnion, a thin, nearly transparent membrane. The embryo measures around 3 mm in length by E15. A balloon-like extension from the caudal region of the embryo is apparent (Fig 5E), likely representing the developing allantois. Between E15 and E17, the embryo grows to a size of around 5 mm in length and a functional circulatory system becomes visible, and heartbeats can be observed (Fig 5F, open arrowhead, S5 File). At E21, the brain vesicles become distinct, and the embryo transitions from a transparent to an opaque appearance and reaches a size of around 13 mm in length (Fig 5G). Limb buds of the forelimbs and hindlimbs are present and begin to take shape (Fig 5G, filled arrowhead). By E27, the embryo reaches a length of 20 mm with a notable increase in head size, developing external ear anlagen and clearly distinguishable eyes forming. The forelimbs and hindlimbs develop digital grooves, and the tail begins to regress (Fig 5H).

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Fig 5. Morphological documentation of embryonic development from E15 to E27.

(A-D) shows the whole gestational sac with the yolk sac containing the embryo oriented to the left, and the placenta oriented to the right. (E-H) shows the embryos after embryonic layer removal, with the head oriented to the left and the caudal region oriented to the right. (E) Filled arrow pointing to the allantois. (F) Open arrowhead pointing to the heart. (G) Filled arrowhead pointing to the hindlimb bud.

https://doi.org/10.1371/journal.pone.0347318.g005

In summary, this protocol enables early and efficient timed mating in guinea pigs and facilitates precise identification and dissection of embryos during the first trimester.

Supporting information

Acknowledgments

We thank Cornelia Cygon, Dr. Nelli Blank-Stein, Dr. Maria Römelt, Dr. Linda Müller, Dr. Nikola Makdissi, Dr. Katharina Mauel, Dr. Eliana Franco Taveras and Theresa Eulgem for their support with guinea pig handling and Dr. med. Metin Cetiner for his help with ultrasound examination.