“Before we can refine, we need to define”

In 2024, around 926,000 animals were bred and about 217,000 animals were imported into Swiss laboratory animal facilities. More than 80 percent of these animals were mice. Accounting for two-thirds, these small rodents also represent the most frequently used animal species in research in Switzerland, mainly in basic research projects.

Despite their central importance, relatively little is known about the health and well-being of female breeding mice. Christina Boyle, group leader at the Institute of Veterinary Physiology of the University of Zurich (UZH), refers to these mouse mothers as “the unsung workhorses of animal research.” She notes that breeding practices are primarily designed to maximize productivity and efficiency.

The idea to study this topic more closely took shape about seven years ago. “Most 3R research has focused on improving welfare for the offspring that later become research subjects,” says Boyle. “But I began to wonder about the mothers. How does the way we breed mice affect their health and well-being?”

According to the UZH physiologist, female breeding mice have rarely been studied in their own right. “We wanted to understand what life is like for breeding females,” she says. “So, we approached this as an exploratory question, curious about how females manage the demands of repeated pregnancy and lactation.” In mice, pregnancy and lactation each last about three weeks, so a full reproductive cycle takes roughly six weeks.

And these reproductive cycles are no small task. Shortly after giving birth, female mice enter a fertile phase known as postpartum estrus. If a male mouse is present, she can mate and become pregnant again almost immediately. This means she is nursing her newborn pups while gestating a new litter at the same time. Since this can happen after each birth, a breeding female may spend several months both pregnant and lactating.

Since October 2022, Boyle has been heading a research project called “Caring for the mothers: the impact of breeding on female mice” within the National Research Programme 79 “Advancing 3R – Animals, Research and Society.” The NRP 79 investigates how to strengthen the 3Rs – the replacement, reduction or refinement of research using animals – in Switzerland. Boyle’s team, which is made up of veterinary medicine and biomedicine students, used a wide range of behavioral and physiological measurements to conduct a large-scale exploratory study examining breeding females after one, two or four consecutive cycles of pregnancy and lactation.

First, they looked at the behavior of the females – how the mother interacts with her pups, and how curious and active she is. “The most experienced mothers are less exploratory. They’re more hesitant to go out and explore new environments,” Christina Boyle says.

Apart from that, the researchers saw no evidence that the number of pregnancies affected maternal behavior. “Even after multiple pregnancies, they’re still good moms. They reliably retrieve their pups and bring them back to the nest,” Boyle says. The team also assessed stress levels using a non-invasive method that measures metabolites of the rodent stress hormone corticosterone in fecal samples. “Again, we saw no difference across one, two or four cycles. These findings suggest that the mothers are not more stressed,” she adds.

The researchers also observed metabolic changes linked to reproduction. They analyzed body weight trajectories, food intake and energy expenditure in the female mice. Similar to what some women experience, mothers with more reproductive experience gained additional weight and retained it for several weeks after weaning their young.

In the next step, the researchers examined whether repeated breeding affects the females’ tissues, including bone mineral density. Because lactating females must supply large amounts of calcium for milk production, the team expected changes in bone structure. Indeed, bone mineral density was reduced. However, most of the loss occurred after the first pregnancy, and it did not substantially worsen with additional births. “The female mouse body clearly responds to the demands of reproduction,” the researcher says. “But the mothers are remarkably resilient. Even with repeated pregnancy and lactation, they continue to care well for their pups. From a welfare perspective, they appear to be doing quite well.”

The results so far indicate that the female mice bred in an academic research setting are healthy, exhibit normal behavior and do not appear to be more stressed. The team is now conducting a follow-up study with mouse mothers from commercial breeding facilities. “This allows us to look at what happens under real-world breeding conditions using retired female breeders,” Boyle says.

In collaboration with ETH Zurich, the researchers are also analyzing the fecal corticosterone levels of the female breeders after different “doses” of reproduction: one to three cycles, four to six cycles, and more than six cycles – so-called superbreeders. These data will reveal whether stress levels increase with greater reproductive load. This new study will also examine the females’ fat mass during peak lactation to determine whether the observed weight gain reflects increased body fat or other physiological changes.

Christina Boyle’s project will officially end in September. What impact could the results have? “The important thing now is to get the data out there, both through publications and by presenting the findings to breeding facilities,” she says. This could help stimulate discussions with the breeders about how breeding practices might be optimized and standardized. One point is particularly important for Boyle – and the NRP 79: “Before we can refine, we need to define. We first needed to establish the baseline and understand the impact of common breeding practices on mouse mothers. Only then can we evaluate whether guidelines or regulations for breeding management should be adapted.”