Background

Maternal exposure to adverse environmental stimuli leads to various physiological changes in the offspring (Hay et al., 2016; Walejko et al., 2019; Battarbee et al., 2020), including predisposition of offspring to the development of obesity and type 2 diabetes (Godfrey et al., 2017; Wesolowski et al., 2018). Physical exercise is highly accessible and a prominent therapeutic tool to combat diet-induced obesity in adults (Peres Valgas da Silva et al., 2019). Epidemiological studies robustly show the beneficial effects of maternal exercise in improving maternal health and neonatal outcomes (Hopkins and Cutfield, 2011; Nascimento et al., 2012; Moyer et al., 2016; da Silva et al., 2017; Rogozinska et al., 2017; Davenport et al., 2018a and 2018b; Ming et al., 2018; Beetham et al., 2019; Hoover and Louis, 2019; Pastorino et al., 2019; Wang et al., 2019). But surprisingly, the biological effects of maternal exercise on offspring health have only been sparsely examined in the context of maternal obesity and high fat diet intake (Laker et al., 2014; Stanford et al., 2015; Stanford et al., 2017; Musial et al., 2019; Uddin et al., 2019), as well as normal chow diet (Raipuria et al., 2015), the effects of maternal exercise of healthy mothers on fetal development remain largely unexplored. Mice is the most commonly used model for biomedical studies; to facilitate mechanistic exploration, an exercise protocol for pregnant mice which can be easily translated into exercise in pregnant women is needed.

Both treadmill exercise and voluntary wheel running are commonly used for exercise training in rodents. We recommend to use treadmill exercise because the exercise intensity and length can be precisely controlled, which is very similar to treadmill training in humans, increasing translational value. On the other hand, large variations in exercise intensity and duration among individual mouse exist in voluntary wheel running, making data interpretation difficult (Goh and Ladiges, 2015). Also, the exercise intensity and duration cannot be controlled in voluntary wheel running and thus difficult to be translated into human exercise. Nonetheless, treadmill exercise training induces low levels of acute stress to mice, but this concern can be alleviated by applying the same procedure to both control and exercised mice. Actually, regular treadmill exercise at moderate intensity has protective effects in stress-related symptoms (Patki et al., 2014; Seo, 2018; Loprinzi and Frith, 2019). Based on exercise guidelines published by American College of Obstetricians and Gynecologists (Zavorsky and Longo, 2011), we propose the following protocol for treadmill exercise, which composes of three stages with exercise intensity calculated based on the maximal oxygen intake (VO₂max) before pregnancy. The initial stage is from embryonic day 1.5 (E1.5) to E7.5, mid-stage from E8.5 to E14.5, and late-stage from E15.5 to birth, with the intensity of exercise at 40%, 65%, and 50% of VO₂max, respectively. Using this protocol, we found that maternal exercise slightly reduced weight gain during pregnancy, but had no effect on birth weight; in addition, offspring metabolic health was improved due to maternal exercise and detailed changes were reported previously (Son et al., 2019 and 2020). Thus, this protocol fits for studying maternal impacts on fetal development and long-term health of the next generation.