PRACTICUM ARTICLE

ANIMAL DEVELOPMENT

Mouse Breeding Techniques for Understanding the Reproductive Process

Nurul Hikmah Utami1*

1
ICP of Biology Education, Group 3, Biology, Makassar State University, Indonesia.

Abstract
Mice, particularly Mus musculus, have played a vital role in animal husbandry and biological research, making them a
key model organism for scientific study. This article delves into the importance and methods of mating mice in
experimental research. The choice of male mice as mating partners is influenced by hormonal stability, while the female's
receptivity is determined by the estrus cycle. A key indicator of successful mating is the presence of a vaginal plug, which
marks the beginning of pregnancy. Various sensory cues guide the mating process, and vocalizations and olfactory
signals also influence mate selection. The experiment "Mating Techniques in Mice" is designed to teach students how to
mate mice successfully, serving as a foundation for future biological and genetic research. This study emphasizes the
importance of timing, proper equipment, and adherence to ethical principles and animal research guidelines. The success
of mouse mating experiments relies on these carefully structured principles, ensuring controlled conditions and valuable
research outcomes.

Keywords: Mice; mouse; mating; experiment..

INTRODUCTION
Research has been used extensively in the efforts to develop technologies in the areas
of animal husbandry and biology. It is necessary to use experimental animals with anatomical
and physiological traits comparable to those of other livestock animals in order to fund these
initiatives. The white mouse (Mus musculus) is one of the most widely used experimental
animals in animal husbandry and biology research. Because they offer benefits over other
experimental animals, mice are utilized in research. The majority (40–80%) of laboratory
animals employed for experimental study are mice (Mus musculus). Mice are extremely
helpful as experimental animals in biological research due to their various benefits, including
their short lifespan, high birth rate, and variety of features. Weight gain serves as a criterion
for measuring growth, a complex process that entails uniform development and simultaneous
increases in both live weight and all body parts (Hasanah & Masri, 2015).
Mice are frequently mated to produce offspring or to conduct teratology or
embryology studies. A fit and healthy person is a suitable candidate for mating since they will
produce descendants or offspring who will likewise be fit and healthy. Male mice can
continue reproducing until they are 14 months old or older after reaching sexual maturity.
Only when the eggs, or ova, are ready to be fertilized, during the estrus season, can female
mice mate with male mice. Copulation can occur between 5 hours before ovulation and up to
8 hours after ovulatio (Nugroho, 2018).
 The decision to choose a male gender is mostly motivated by the fact that male mice
either lack or have very little estrogen hormones. Male mice have more stable hormonal
conditions than female mice because female mice experience hormonal shifts during certain
times, such as during the estrus cycle, pregnancy, and lactation, which can have an impact on
the test animals' psychological health. Furthermore, compared to male mice, female mice
often exhibit higher stress levels, which may interfere with the testing procedure (Yusuf et
al., 2022).
Mating mice involves placing female mice with male mice in the same cage using the
one-mating method. To determine whether a female mouse has been mated, the presence of a
vaginal plug is observed on the following day after mating. The vaginal plug is a clump of
gelatin that serves to prevent the spilling of spermatozoa. If a vaginal plug is present, it is
considered as evidence of copulation, and this day is designated as Day 0 of pregnancy. Once
pregnancy is confirmed, the female mouse is moved to an individual cage. Each mouse is
identified by marking its ear and recording the date of estrus, date of mating, date of DEET
administration, and the date of sacrifice (Kusmawati, 2016).
Making mating decisions requires the gradual integration of various sensory cues. A
mouse that comes into contact with a conspecific of the opposite sex will exhibit a series of
stereotypical mating behaviors. If both parties are sexually receptive, they will exhibit
sexually appetitive behaviors during this interaction. A male mouse will investigate his
possible mate, especially in the anogenital region, and will follow and pursue a female. When
a female mouse approaches a male repeatedly and makes darting motions, she is indicating
that she is motivated by lust (Karigo & Deutsch, 2022).
The gregarious house mouse (Mus musculus) uses chemical and auditory cues to
communicate with other mice in its social group. Ultrasonic vocalizations (USVs) are made
by male mice, and they may be used to woo females. Significant variability is shown by these
male USVs, which vary between species and between mice, possibly indicating unique
individual identities. Interestingly, female mice display a preference for USVs from
unfamiliar males of their own species. However, this preference can fluctuate based on
previous exposure to male odors, such as soiled bedding, and the female's estrus state. Female
mice are particularly attracted to male urine containing a high level of the protein known as
darcin. Notably, the number of male USVs and the concentration of darcin in urine seem to
be influenced by the functioning of the male's immune system. Collectively, these findings
imply that female preferences for male signals are not just influenced by attraction to male
USVs on their own. Rather, it seems that female preferences are shaped through an
interaction between olfactory signals and USVs. The extent to which males react to these
multimodal female signals has, however, received comparatively little research (Ronald et al.,
2020).
The goal of the "Mating Techniques in Mice" experiment is to teach students how to
mate mice successfully. The method of mating mice is an essential component of biological
and genetic research in a scientific setting because it offers profound insights into genetic
inheritance and trait transmission. Students will learn about the technical steps in mouse
mating through this experiment, including proper mate selection, proper mating techniques,
as well as observation and analysis of the experiment's results. Students can use a thorough
understanding of mouse mating behaviors in their research and future development in the
biological and genetics domains. As a result, this experiment has a significant impact on how
students understand the fundamentals of biology.
METHODS
In the experiment on breeding mice, the choice of 6:00 PM for the mouse mating
process is not solely based on the time aspect within a day, but is also carefully considered to
allow better monitoring of the mice's conditions, meticulous preparation, and to provide
adequate lighting for effective visual observation during the mating process. This makes the
selection of this timing a strategic choice in the effort to achieve accurate and successful
results in this experiment.
Continuing with the experiment on breeding mice, apart from the previously
discussed timing, the tools and materials used also play a crucial role in ensuring the success
of the experiment. The tools used include mouse cages, water bottles, identification cards,
and paper clips. Mouse cages are vital in ensuring the separation and supervision of the
mouse pairs to be mated, while water bottles are used for providing drinking water.
Identification cards and paper clips are useful for identifying and labeling mice during the
experiment. Materials such as mouse feed, which can be rice bran, and tap water are used to
ensure appropriate conditions and nutrition during the mouse mating process, making them
essential factors in achieving credible experimental results. All these elements are
interconnected and play a role in carrying out the experiment carefully and systematically.
Continuing the implementation of the experiment on breeding mice, the procedures
carried out are highly detailed and play a crucial role in the success of this experiment. The
initial stage involves preparing the mouse cages, where two cages lined with approximately 2
cm of rice bran must be prepared and changed every 3 days. The selection of mice ready for
mating is done by considering a minimum age of 10 weeks, as well as signs such as swelling
of the vulva and a change in color to reddish in female mice. Female and male mice are then
placed in the same cage in the evening, around 6:00 PM. The cages are located indoors with
lighting set for 12 hours of light (from 6:00 AM to 6:00 PM) and 12 hours of darkness (from
6:00 PM to 6:00 AM), and room temperature maintained in the range of 22.5 to 24.5 degrees
Celsius.
Next, on the following day, an examination of the vaginal plug in the mice is
conducted. The success of mouse mating is indicated by the presence of a vaginal plug, and
this day is marked as day 0 of pregnancy. All crucial information regarding each mouse is
meticulously and accurately recorded on identification cards corresponding to the purpose of
further mouse maintenance. These identification cards are then attached to the mouse cages to
facilitate systematic monitoring and labeling throughout the duration of the experiment. All
of these steps reflect a high level of precision and seriousness in conducting the experiment,
ensuring that the data generated is valid and reliable within the research context.

RESULTS
Tabel 1. Results of the Laboratory Activity
No. Documentation Description
1. Preparation for Mouse Mating All the necessary tools and materials are
prepared.
 Both sexually mature mice and the vulva of
female mice appear slightly swollen and
reddish.

2. Mouse Mating Male and female mice are placed in the same
cage at 6:00 PM.

Examination of vaginal plugs is conducted at

6:00 in the morning.

Mice with vaginal plugs are placed in

separate cages.
 Mouse identification cards are filled out and
hung on the mouse cages.

DISCUSSION
The success of the mouse breeding experiment can be attributed to the visible
presence of vaginal plugs. These plugs, observed in the female mice, indicate successful
mating. The appearance of vaginal plugs serves as a reliable and concrete indicator of the
effectiveness of the breeding technique employed in this experiment. This clear and
unmistakable sign ensures that the experiment's objectives have been achieved, contributing
to the overall success of the study on mouse breeding techniques.
After a male has successfully pursued and motivated a female, he will attempt to
mount her from the rear, grasping her flanks with his forepaws and displaying pelvic
thrusting movements. If a female is sexually receptive, she will exhibit readiness behaviors,
including lordosis, which involves arching her lower back toward the floor. If a female is not
receptive, she will display rejection behaviors, such as attempting to escape, kicking, and
assuming a defensive posture to avoid the male's mating attempts, often accompanied by
emitting broad-frequency vocalizations (squeaks) when the male attempts to mount. Once a
male has successfully mounted a willing female, he will proceed with intromission at a
slower thrusting pace and deeper movements, which can be visually distinguished from the
thrusting performed during mounting. A varying number of mounts and intromissions will be
conducted until ejaculation is achieved. After a male completes copulation through
ejaculation, he engages in post-copulatory grooming of his genital area. Following
ejaculation, the male enters a refractory period during which he is not attracted to the same
willing female for at least 24 hours (Karigo & Deutsch, 2022).
Experiments involving the mating of mice begin with the selection of pairs of mice
with specific characteristics or genes to be studied. These pairs are placed together in a clean
and sterile cage equipped with shelter, food, and water. The timing of mating must be
carefully chosen to align with the mice's reproductive cycle. The entire process must be
conducted ethically, adhering to applicable animal research guidelines, and must serve a
beneficial scientific purpose.
The equipment used in mouse mating experiments serves several crucial functions in
conducting the experiment. The initial step involves selecting pairs of mice with specific
characteristics or genes to be studied. The clean and sterile cage functions as the environment
where the mouse pairs are placed for mating and the subsequent rearing of their offspring.
The cage plays a vital role in ensuring cleanliness to reduce the risk of infection or
contamination that could affect the experiment's outcomes. Additionally, the cage provides
protection for the mice, giving them a necessary shelter during the mating and pregnancy
processes. The provision of food and water within the cage is essential for maintaining the
health and well-being of the mice throughout the experiment. Furthermore, the careful timing
of mouse mating in line with their reproductive cycle is critical to the experiment's success.
This equipment also ensures that the entire experimental process is conducted ethically,
following applicable animal research guidelines, safeguarding the welfare of the animals, and
generating scientifically valuable data. Overall, this equipment aids in creating controlled
conditions and ensures that the experiment aligns with the set research objectives.
The mouse cage serves as the environment where mouse pairs are placed for mating
and the subsequent rearing of their offspring. The cage is essential for maintaining cleanliness
and sterility while providing shelter, food, and necessary water sources. The drinking bottle
functions as a water supply for the mice, ensuring their hydration and overall health during
the experiment. Identification cards are used for labeling and tracking individual mice
involved in the experiment, enabling researchers to distinguish and uniquely identify each
mouse. Paper clips, while not directly related to the mice, may be used for data recording or
as aids in observing mouse behavior or experiment outcomes. Altogether, these tools are used
to create controlled conditions, ensure the success of the experiment, and enable accurate
monitoring.
The principles behind the equipment used in mouse mating experiments can be
outlined as follows. The initial step involves selecting specific pairs of mice with desired
characteristics or genes for study, with the principle of achieving research objectives through
appropriate pair selection. The clean and sterile cage serves to create an environment free
from contamination and infection, adhering to the principle of maintaining cleanliness to
minimize external disturbances that could affect experiment outcomes. Moreover, the cage
provides protection for the mice during mating and pregnancy, adhering to the principle of
providing a safe and comfortable space for interaction and offspring birth. Monitoring the
mice's reproductive cycles and carefully selecting the timing of mating aligns with the
principle of maximizing the experiment's success in achieving pregnancy and offspring birth.
Additionally, providing food and water within the cage adheres to the principle of ensuring
mice have access to necessary nutrition and hydration, supporting their health and well-being.
Ethical considerations and adherence to applicable animal research guidelines are
fundamental principles underpinning the entire process, with the goal of safeguarding animal
welfare and ensuring the experiment is conducted in an ethical and scientifically rigorous
manner. Collectively, these principles work together to establish controlled conditions and
ensure that the experiment aligns with the research objectives set forth.
In the overall discussion above, there is a consistent set of principles underlying
mouse mating experiments. Starting from the careful selection of mouse pairs to the pivotal
role of clean and sterile cages in creating an ideal environment for mating and offspring
rearing, everything is structured with the goal of achieving valid research outcomes.
Monitoring the reproductive cycles of mice and timing mating appropriately becomes crucial
aspects to maximize the experiment's success, while the provision of food and water supports
the health and well-being of the mice throughout this process. Ultimately, the principles of
ethics and adherence to animal research guidelines provide a strong moral foundation,
ensuring animal welfare and that the experiment is conducted ethically and in accordance
with scientific standards. All of these principles work together to establish controlled
conditions and ensure that the experiment aligns with the research objectives set, resulting in
valuable and reliable data.
CONCLUSION
For mouse mating experiments, it's crucial to consider a range of important principles
and steps. Central to achieving research objectives is the careful selection of appropriate
mouse pairs. Clean and sterile cages play a pivotal role in maintaining controlled conditions,
preventing infections, and ensuring the well-being of mice throughout the mating and
pregnancy process. The close monitoring of the mice's reproductive cycles and precise timing
of mating are critical factors in determining the success of the experiment. Additionally,
providing sufficient food and water is essential for sustaining the health of the mice. The
entire process must adhere to ethical principles and established animal research guidelines to
safeguard animal welfare and maintain the scientific integrity of the research.
In short, ensure the careful selection of mouse pairs, prioritize the welfare of the
animals, and adhere to ethical principles and relevant animal research guidelines to guarantee
valid and valuable research outcomes.

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