Swimming Exercise Protocol and Care Methods for Pregnant Rats

Summary

Here, we present a protocol to delineate rat breeding methods, swimming training procedures, and post-breeding nursing protocols for pregnant rats after training. This protocol provides an animal model for studying the effects of maternal exercise during pregnancy on offspring and its underlying mechanisms.

Abstract

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, cardiovascular disease, and cancer. Studies using animal models have investigated how maternal factors such as undernutrition, overnutrition, obesity, and exposure to chemicals or hypoxia affect fetal development and offspring health, leading to issues like low birth weight, high blood pressure, dyslipidemia, and insulin resistance. Given the increasing prevalence of overweight and obesity among reproductive-age women, effective interventions are critical. Maternal exercise during pregnancy has emerged as a key intervention, benefiting both mother and offspring and reducing the risk of disease. This study compares the differences of three exercise models on pregnant rats: voluntary wheel running, motorized treadmills, and swimming. Swimming is the most beneficial option due to its safe and controlled intensity levels. This protocol details the rat breeding methods, swimming training during pregnancy, and post-breeding nursing protocols. This model, suitable for various rat and mouse species, is useful for studying the benefits of maternal exercise on offspring health and intergenerational wellness.

Introduction

The Developmental Origins of Health and Disease (DOHaD) concept emerged over the last two decades, which has shown that environmental influences during early development affect the risk of later pathophysiological processes associated with chronic non-communicable disease (NCD), especially diabetes, cardiovascular disease, and some types of cancer¹. During the most plastic phase of fetal development, where offspring are exposed to the intrauterine environment, the gene and environment interaction and uteroplacental perfusion are crucial factors of fetal reprogramming². Previous studies have predominantly utilized animal models to investigate the detrimental effects, such as maternal undernutrition, overnutrition, or obesity during pregnancy, and prenatal exposure to chemicals or hypoxia, on fetal development and long-term phenotypic effects in the offspring³. This produced low birth weight and later growth and produced adverse effects on offspring, including elevated blood pressure, dyslipidemia, and insulin resistance⁴. Since the rate of overweight and obesity is rapidly increasing among women of reproductive age, establishing effective interventions to prevent the intergenerational transmission of these deleterious maternal disorders is urgently needed, which has an important impact on population health^5,6.

Exercise has long been recognized as an important preventive therapeutic for type 2 diabetes, hypertension, and several other diseases⁷. Exercise during pregnancy has beneficial effects for the mother and confers beneficial effects on offspring, thus reducing the maternal transmission of disease to offspring⁸. Several years of research have demonstrated exercise's safety and profound benefits, including substantial reductions in common pregnancy conditions such as gestational diabetes mellitus^9,10. Regarding maternal and fetal safety, conducting exercise training and post-nursing protocols is challenging.

David¹¹ et al. described animal exercise models applicable to cardiovascular health research. The three commonly used exercise models, voluntary wheel running, motorized treadmills, and swimming, have advantages and disadvantages. The voluntary wheel running closely mimics the locomotor behavior of rats, enabling them to move according to their circadian rhythm. However, this sort of exercise needs more precise regulation of intensity and duration. The motorized treadmill can control exercise intensity, volume, and duration, but sometimes it needs electric stimulation, which may trigger physical and psychological stress in the animals. Swimming is widely used in rodent studies due to the inherent swimming ability of rats, which can also avoid electric stimulation or mechanical damage to their feet and tails¹². In studies on prenatal exercise models^13,14, swimming is a commonly used exercise modality. As pregnancy progresses and the abdomen of the rat swells, treadmill and wheel running exercises may cause repeated contact of the abdomen with hard surfaces. In contrast, the water environment in swimming provides buoyancy, reducing the risk of exercise-related injuries for pregnant animals.

This protocol presents the rat breeding methods, how to perform the swimming training during pregnancy, and the post-breeding nursing protocols for pregnant rats after training. This pregnant swimming training model can be applied to a wide range of rat and mouse species, which may thus provide a useful rodent model for future investigation using animal-based models to study the mechanisms regulating the beneficial effects of maternal exercise on offspring health and the benefits of exercise across generations.

Protocol

All methods described here have been approved by the animal ethical committee at Beijing Sport University and carried out in compliance with the National Institutes of Health (NIH) guidelines and the Chinese animal protection laws and institutional guidelines.

1. Mating and feeding rats

1. Purchase specific pathogen-free (SPF) rated 10-week-old Wistar female rats and 11-week-old Wistar male rats.
2. Implement adaptive feeding: Feed the Wistar rats ad libitum with a standard rodent diet and provide access to tap water for one week in the designated feeding facility. Ensure all rats are housed in a 12-h light-dark cycle at a constant temperature of 22 °C and humidity of 45-55%.
3. Examine the estrous of the female rat.
   1. Prepare the equipment and materials, including clean glass microscope slides, 2 mm diameter cotton swabs, saline solution (0.9% NaCl), and microscope.
   2. Label each microscope slide with the date and rat identification.
   3. Between 6 and 7 PM, to minimize stress, restrain the female rat gently. Grasp the base of the tail with the thumb and forefinger while applying pressure from the other fingers onto the lumbar vertebra of the rat.
   4. Lift the tip of the tail and expose the vaginal. Moisten a cotton swab with saline solution, then put the tip into the vagina and gently roll the tip to collect vaginal secretions.
   5. Roll the swab tip across the slide, avoiding reapplication to the same area.
   6. Examine the smear under the microscope and use the low magnification objective (10x) to locate and observe the cells.
      NOTE: During estrus, the vaginal secretions have larger epithelial nuclei vanished, replaced by squamous exfoliated keratinized epithelial cells that accumulated into a mound.
   7. Record the stage of the estrous cycle for each rat.
4. Between 7 and 8 PM, place a male and a female rat in the mating cage.
   NOTE: The female rats need to be in proestrus or estrus for mating.
5. On the second day, between 7 and 8 AM, inspect the mating cage for copulatory plugs. If a copulatory plug is found at the base of the mating cage, proceed to create a vaginal secretion smear for the female rat following the same method as described in steps 1.3.4- 1.3.6.
   NOTE: Copulatory plugs are milky white or yellowish gelatinous materials made of male rat seminal vesicle and prostate secretions combined with vaginal secretions.
6. Confirm pregnancy in the female rat: after finding copulatory plugs in the mating cage, observe sperm on the vaginal secretion smear. Designate this day as gestation day 1 (GD1).
7. Isolate the pregnant rat in a separate cage.
   NOTE: During the initial stages of pregnancy, it is imperative to prevent a pregnant rat from encountering unfamiliar male chemical cues in its environment or from cohabiting with an unfamiliar male rat.
8. Rat gestation periods typically last 21-23 days. Measure and record the rats' body weights from GD1 to GD20.
9. On GD20, replace the pregnant rat's cage with a clean one and place medical absorbent cotton (similar in size to the rat) in the cage. Supply sufficient water and food for seven days' survival, and position the cage in a quiet location.
   NOTE: Do not change the cage for at least 1 week after rat parturition. When switching to a clean cage, transfer the used absorbent cotton from the dirty cage into the new one to ensure the rats can recognize the scent of their offspring.
10. On the 21^st day postpartum, separate the offspring by gender and place them into two cages.
    NOTE: Due to the smaller size of the 21-day-old offspring, place the hard feed pellets in a glass petri dish inside the cage.

2. Prenatal swimming training program

1. Conduct adaptation training for all experimental female rats for 5 days.
   1. Prepare a transparent plastic container at least 20 cm in height and fill it with 10 cm of water at 34 ± 1°C.
   2. Grasp the tip of the rat's tail and lift it, then place the rat in the water.
   3. During the 15-min adaptive training, closely monitor the water temperature every 5 min, adding warm water to maintain the temperature.
   4. After the training, transfer the rat to clean water at 34 ± 1°C to wash its fur. Then, transfer the rat to a cage with towels and use a hairdryer to dry its fur.
      NOTE: Noise and a hot environment can cause stress to rats. Therefore, it is recommended to use a temperature-adjustable and quiet hair dryer.
   5. Kindly return the rat to its respective cages.
2. Rat mating (follow the steps 1.3-1.6).
3. After step 1.7, randomly assign pregnant rats into a sedentary group (SED) and an exercise group (EX).
4. Weigh the rat daily before every training session.
   NOTE: During the adaptive training, the weight loss of rats should not exceed 3 g/day. During the prenatal training, the weight of the rats should gradually increase. If the rats lose more than 3 g per day, reduce the daily training time by 50% to allow for recovery.
5. Conduct prenatal swimming training for the EX from GD1 to GD20, 6 days per week, from 8:00 to 9:00 in the morning.
   1. Prepare a circular water bucket with a diameter of 50 cm and a minimum height of 50 cm and fill it with 40 cm of water at a temperature of 34 ± 1°C.
   2. From GD1 to GD5, initially perform the swimming training for 20 min a day and progressively increase to 60 min a day.
   3. From GD6 to GD20, continue the exercise training for 60 min per day for 6 days per week.
   4. For aftercare, refer to steps 2.1.4-2.1.5.
   5. After exercise, give the EX rat 5 g of rodent sunflower seeds to provide nutritional support.
6. During the EX training every day, place the SED rat in the same water environment as adaptive training (step 2.1.1). Let the SED rat spend the same time in the water environment as the EX rat. After exercise, give the SED rat 5 g of rodent sunflower seeds to provide them with nutritional support and soothing emotions.
7. After each swim training session, clean and disinfect all equipment with ultraviolet light, including water buckets and towels.

3. Physical characteristics of fetuses

1. On the morning of GD20.5, anesthetize the female rat with 3% isoflurane and euthanize it in a CO₂ chamber.
2. Use a surgical knife to make an incision in the lower abdomen of the rat. Make sure that the incision slants towards both sides of the abdomen, ensuring complete exposure of the uterus.
3. Carefully open the uterus using forceps and scissors to expose the fetuses and placentas. Gently separate the thin amnion surrounding the fetuses using forceps to avoid scissor-induced damage to the fetuses and placentas.
4. Clean the bodies of fetuses using sterile gauze to remove blood and mucus. Sequentially separate fetuses and placentas by their anatomical positions, then weigh them.
5. Observe the distance between the anus and genitalia to determine the gender of the fetuses. Male fetuses have a greater distance than females. Record the gender and quantity of fetuses for future reference.
6. Measure the length of the fetuses using a caliper while lying flat. Because the fetuses are still alive, they may curl up after being dissected, so unfold their bodies to take measurements.
   NOTE: To ensure the consistency of experimental results and the measurement methods are consistent, the same experimenter maintains the same standards throughout. Fetuses were euthanized by institutional recommended guidelines and tissue samples were collected for further analysis as needed.

Results

In the rat breeding methods section (Figure 1), we determine the estrous cycle of female rats by making vaginal secretion slides to increase the success rate of breeding. The rats' estrous cycle lasts 4-5 days, including proestrus, estrus, metestrus, and diestrus. Female rats in metestrus or diestrus exhibit behaviors such as distancing themselves from males. At the same time, those in estrus are willing to mate, and ovulation in female rats mostly occurs at the end of estrus. During proestrus, vaginal smear samples primarily contain nucleated epithelial cells with occasional keratinized cells. In estrus, the vaginal smear predominantly consists of keratinized squamous epithelial cells clumped together in piles, with the disappearance of the enlarged nuclei of epithelial cells seen in metestrus. In metestrus, the vaginal smear mainly contains white blood cells with a few keratinized epithelial cells. The diestrus stage is characterized by a predominance of white blood cells, with occasional nucleated and keratinized epithelial cells (Figure 1A).

The copulatory plug is a gel-like substance composed of vaginal and seminal secretions. The female rat forms a copulatory plug to prevent other male rats from mating again while also blocking the female rat's vagina to prevent the male rat's sperm from flowing out. The presence of the copulatory plug does not necessarily indicate successful breeding but rather only signifies the occurrence of mating behavior. Therefore, it is necessary to perform vaginal smear slides on the female rat again in the morning after mating. The presence of sperm in the vaginal smear indicates successful breeding. It is important to monitor the weight of female rats after this day to observe if it increases gradually, which is a sign of successful pregnancy. If there is no copulatory plug formation within two days of mating, or if a copulatory plug forms but no sperm is observed in the smear, it is recommended to replace the male rat and attempt breeding again. By examining vaginal secretion slides under a microscope, we determine the stage of the estrous cycle in female rats and use those in proestrus and estrus for breeding. Pregnant rats were randomly assigned to a sedentary (SED) or exercise (EX) group, with swimming training conducted from GD1 to GD20 (Figure 1B). To observe the copulatory plugs formed after rat mating, we use a special designed mating cage for rat mating. The mating cage consists of a base tray, a wire mesh bottom, a cage frame without a bottom, and a wire cage cover (Figure 1C).

Before giving birth, pregnant rats may display nesting behavior (Figure 1E), utilizing materials in their environment to construct a cozy and warm nest for their offspring. It is recommended to place an amount of defatted cotton in the cage corresponding to the size of the pregnant rat whenever transferring them to a new clean cage on GD20. To study young rats, they must be raised until a certain age. Rats are weaned at about 21 days after birth. Therefore, on the 21st day after birth, baby rats are separated by gender (Figure 1F) for individual rearing. The method of identifying the gender of rats involves observing the distance between the genitalia and the anus. In males, the distance is greater, and they have fur between the genitalia and the anus. Male genitalia are more prominent and pronounced compared to females. Female rats have more noticeable nipples than males.

Daily weighing and recording from GD1 to GD20 is recommended during rat pregnancy (Figure 2). Rat weight should gradually increase during pregnancy (Figure 2A), and daily weights should be compared to the previous day's weight. This is crucial because rats tend to consume their embryos following a miscarriage, making it difficult to detect through observation alone. Additionally, monitoring rat weight can help identify excessive exercise. If the rat's weight decreases by more than 3 g, reduce the duration of exercise training for that day by 50%. On GD20.5, pregnant rats are dissected to obtain fetuses and placentas, and the ratio of fetal weight to placental weight is defined as placental efficiency (Figure 2B). Measure the length of the fetus from the tip of the head to the bottom of the rump in a lateral position using a ruler to obtain the fetal body length (Figure 1G and 2C).

Figure 1: Rat pregnancy model. (A) The vaginal smear results at different estrous periods, rats' copulatory plug images, and sperm presence in vaginal smears on the second day after mating. (B) Schematic illustrating the prenatal swimming training program. (C) Overall views of the mating cage for the rats. (D) A photo of swimming training for pregnant rats demonstrating the diameter of the water bucket used. (E) The nest-building behavior of rats. (F) The different reproductive organ appearances in male and female rats at 21 days of age. (G) The appearance of fetuses and placentas and a schematic representation of the method for measuring fetus body length. Please click here to view a larger version of this figure.

Figure 2: The pregnant rats' body weight and the fetuses' physical characteristics. (A) The weight changes of pregnant rats from GD1 to GD20 in the SED and EX groups (n = 9 each). (B) The difference in placental efficiency between the two groups. (C) The variation in body length of fetuses between the SED and EX groups. Please click here to view a larger version of this figure.

Figure 3: Comparison of different exercise methods for pregnant female rats. The advantages and disadvantages of different exercise methods for pregnant female rats: voluntary wheel running (VWR), motorized treadmill (MT), and swimming. Please click here to view a larger version of this figure.

Figure 4: Swimming exercise program. (A) The complete process of mating, pregnancy, swimming exercise, parturition, and lactation described in protocol steps 1.1-1.10 using female Wistar rats. (B) The process of swimming training during pregnancy described in protocol steps 2.1-2.6, including adaptation training and formal training. The water temperature is maintained throughout the process at 34 ± 1 °C. During adaptation training, the water depth is 10 cm, while during formal training, it is 10 cm for the SED and 40 cm for the EX. Please click here to view a larger version of this figure.

Table 1: Possible problems in swimming exercise and postpartum care for pregnant rats and their solutions. Please click here to download this Table.

Table 2: A comparative analysis of exercise programs in pregnant female and male rats or mice. MT: motorized treadmill; VWR: voluntary wheel running; MLC: maximum load capacity test. Please click here to download this Table.

Discussion

This study aims to establish a feasible exercise program for pregnant rats, including detailed rat mating methods, swimming training protocols, and research methods to evaluate physiological indicators in fetuses. We have developed a prenatal swimming exercise training model through practice, with corresponding solutions to potential issues that may arise in the protocol (Table 1). This protocol will make it more practical and assist researchers in establishing prenatal exercise models.

Our research results indicate that using the rat pregnancy swimming training program we designed does not interfere with the normal pregnancy process of rats and does not lead to adverse pregnancy outcomes. The impact of prenatal exercise on pregnancy outcomes in rats was evaluated by measuring miscarriage rates and litter size. The study concluded that prenatal exercise did not significantly affect these factors^15,16. To study fetuses, researchers dissect pregnant rats on GD20.5 to obtain fetal samples. During dissection, fetuses and placentas are collected, and it is crucial to track the number of offspring per litter to determine pregnancy outcomes, requiring tallying of fetal numbers and fetal body length. Additionally, placental efficiency is the ratio of fetal weight to placental weight, necessitating measurements of both fetal and placental weights. Our previous findings indicated that prenatal exercise did not significantly affect fetal body length, but it significantly improved placental efficiency in the hypertensive exercise group compared to the hypertensive control group¹⁵. Maternal behavior^13,17 can serve as an indicator for evaluating the impact of exercise during pregnancy on the mother. Therefore, indicators such as the miscarriage rate, litter size, fetal body length and weight, placental efficiency, and maternal behavior can be utilized for evaluating the effects of gestational exercise on fetal outcomes. Therefore, we recommend adopting our experimental protocol when shaping pregnancy exercise models to avoid unnecessary harm to experimental animals.

Since the development of the DOHaD theory, there has been a greater emphasis on exploring the influence of early-life environmental variables on individual health¹⁸. Numerous studies have been conducted to investigate putative pathophysiological pathways in fetal-originated disorders using animal models¹⁹. Because rodent models that include exercise during pregnancy may successfully imitate this process, various types of exercise are being explored in animal models with diverse fetal-derived illnesses²⁰. Autonomous running wheels, running platforms, weight-bearing climbing equipment, and swimming protocols are examples of common workout techniques. Obesity, diabetes, and hypertension are all typical animal models²¹. We can see that different exercise routines have distinct advantages and drawbacks from classifying and comparing the exercise methods used in various research studies^{15,16,21,22,23,24} (Figure 3 and Table 2).

We offer a detailed explanation of rat pregnancy control, execution of the swimming exercise routine, and assessment of fetuses and progeny in this protocol. By carefully controlling the mating behavior of rats, it was possible to get a more accurate estimate of how long a pregnancy lasts. This enabled exercise interventions during pregnancy and created a model for studying prenatal exercise. At 21 days of gestation, fetuses and placenta samples can be dissected to evaluate the intergenerational effects of maternal activity during pregnancy on placental function and fetal development. Furthermore, the offspring of gestational exercise rats were fed until adulthood and even old age to study the long-term physiological and pathological processes underlying these effects.

When setting up a swimming training program for rats, it is important to consider that they may try to avoid swimming by hiding in corners, climbing out of the pool, holding onto other rats, or even floating on air bubbles in their fur. To prevent this, the swimming pool should be made of plastic and be cylindrical. The water in the pool should be deeper than 1.5 times the length of the rat, measured from its nose to the end of its tail. The depth of the water should primarily be over 50 cm, and the surface area should be large enough to allow for unlimited swimming. Providing rats with a minimum swimming space of 1000 cm² is important¹¹. Therefore, we have designed a swimming training apparatus that consists of a bucket with a height of 55 cm and a diameter of 50 cm. During swimming training, the water level should be 40 cm, and the water surface diameter should be 45 cm. Additionally, it is important to maintain the water temperature at 34 ± 1°C. This apparatus is suitable for one rat to train at a time. It is important to gradually increase the intensity of swimming training for pregnant rats. This allows them to adapt to the exercise and aquatic environment. Sudden changes in intensity could cause stress reactions, which may increase the risk of miscarriage. Also, it is recommended to have at least one rest day per week to prevent exercise-induced fatigue.

The animal models used in our previous research²⁵ on exercise during pregnancy included spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rat controls, making this protocol's exercise routine equally suitable for hypertensive rats or other disease models. Our study already found that exercise during pregnancy significantly reduced blood pressure in 3-month-old hypertensive offspring and that prenatal exercise had an important impact on reducing cardiovascular reactivity¹⁴. It was also discovered that maternal exercise induces epigenetic modifications of L-type voltage-gated Ca²⁺ channel α1C (Cacna1c) gene, angiotensin II type 1 receptor (Agtr1a) gene and NADPH oxidase-4 (Nox4) gene to improve vascular function in hypertensive offspring^14,25,26.

Multiple factors may affect the reproductive process in rats. Rat age, health state, sperm motility, female rat estrus, temperature, humidity, and light shifts in the breeding habitat influence mating. Furthermore, indoor noise and vibrations during breeding may cause agitation in hypertensive rats, lead to miscarriage, or reject postpartum feeding. Soundproof cotton covers the animal cages during breeding to reduce these impacts.

In this protocol, we offer a comprehensive guide on rat mating methods, a swimming exercise program for pregnant rats, postnatal care for pregnant rats, methods for raising offspring rats, and observational techniques for measuring the physical characteristics of fetuses. Our research findings contribute to studying the intergenerational inheritance of maternal exercise and demonstrate a rational and effective research approach. We aim to establish an effective swimming exercise model for pregnant women within the field of exercise physiology, which will aid future research in this area.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Caliper	Mitutoyo Measuring Instruments (Shanghai) Co., Ltd	530-101 N15	Fetus dissect
Circular water bucket	Naliya	N/A	Rat swimming
Experimental Surgical Instrument	Shenzhen RWD Life Technology Co., LTD	SP0001-G	Fetus dissect
Glass Microscope Slides	Jiangsu Shitai experimental equipment Co., LTD	80312-3161	Vaginal smear
Glass petri dish	Merck Life Technologies	BR455751-10EA	Offspring rearing
Hairdryer	Panasonic	EH-WNE5H	Post-swimming care
Information Card	Zhongke Life Science	SS3	Rat mating
Isoflurane	Shenzhen RWD Life Technology Co., LTD	R510-22-10	Fetus dissect
Light microscope	Olympus	IX71-F22PH	Vaginal smear
Mating cage	Zhongke Life Science	SS3	Rat mating
Medical Absorbent Cotton	Hongxiang Sanitary Materials Co., LTD	N/A	The pregnant rats are anticipating giving birth
Rodent Anesthesia Machine, Gas Anesthesia	Shenzhen RWD Life Technology Co., LTD	R500IE	Fetus dissect
Rodent breeding feed	Beijing Huafukang Biotechnology Co., LTD	1032	Pregnant rat feeding
Rodent maintenance feed	Beijing Huafukang Biotechnology Co., LTD	1022	Offspring rearing
Rodent sunflower seeds	Jolly	JP241	Nutritional supplement
Soundproof cotton	Kufu Medical Instrument	N/A	The pregnant rats are anticipating giving birth
Sterile Cotton Swab (2 mm diameter)	Kufu Medical Instrument	N/A	Vaginal smear
Sterile gauze	Kufu Medical Instrument	N/A	Fetus dissect
Stroke-physiological Saline Solution (0.9% NaCl)	Shandong Hualu Pharmaceutical Co., LTD	N/A	Vaginal smear
Thermometer	Beekman organism	N/A	The monitoring of rat swimming
Towels	Grace	N/A	Post-swimming care
Transparent plastic container	Naliya	N/A	Swimming adaptive training
Ultraviolet light	Merck Life Technologies	Z169633-1EA	Post-swimming care
Water heater	Haier	EC6001-Q6S	Rat swimming
Weight Scale	Electronlc Acale	JM.A10001	Body weight measurement
Wistar Rats	Vital river Laboratory Animal Technology Co., LTD	N/A	Experiment