This work was supported by the National Natural Science Foundation of China (32071174, 32200941, 32371183, and 31771312).

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
<ol>
	<li>Purchase specific pathogen-free (SPF) rated 10-week-old Wistar female rats and 11-week-old Wistar male rats.</li>
	<li>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 &#176;C and humidity of 45-55%.</li>
	<li>Examine the estrous of the female rat.
	<ol>
		<li>Prepare the equipment and materials, including clean glass microscope slides, 2 mm diameter cotton swabs, saline solution (0.9% NaCl), and microscope.</li>
		<li>Label each microscope slide with the date and rat identification.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Roll the swab tip across the slide, avoiding reapplication to the same area.</li>
		<li>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.</li>
		<li>Record the stage of the estrous cycle for each rat.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Confirm pregnancy in the female rat:&#160;after finding copulatory plugs in the mating cage, observe sperm on the vaginal secretion smear. Designate this day as gestation day 1 (GD1).</li>
	<li>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.</li>
	<li>Rat gestation periods typically last 21-23 days. Measure and record the rats&#39; body weights from GD1 to GD20.</li>
	<li>On GD20, replace the pregnant rat&#39;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&#39; 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.</li>
	<li>On the 21st 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.</li>
</ol>
2. Prenatal swimming training program
<ol>
	<li>Conduct adaptation training for all experimental female rats for 5 days.
	<ol>
		<li>Prepare a transparent plastic container at least 20 cm in height and fill it with 10 cm of water at 34 &#177; 1&#176;C.</li>
		<li>Grasp the tip of the rat&#39;s tail and lift it, then place the rat in the water.</li>
		<li>During the 15-min adaptive training, closely monitor the water temperature every 5 min, adding warm water to maintain the temperature.</li>
		<li>After the training, transfer the rat to clean water at 34 &#177; 1&#176;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.</li>
		<li>Kindly return the rat to its respective cages.</li>
	</ol>
	</li>
	<li>Rat mating (follow the steps 1.3-1.6).</li>
	<li>After step 1.7, randomly assign pregnant rats into a sedentary group (SED) and an exercise group (EX).</li>
	<li>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.</li>
	<li>Conduct prenatal swimming training for the EX from GD1 to GD20, 6 days per week, from 8:00 to 9:00 in the morning.
	<ol>
		<li>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 &#177; 1&#176;C.</li>
		<li>From GD1 to GD5, initially perform the swimming training for 20 min a day and progressively increase to 60 min a day.</li>
		<li>From GD6 to GD20, continue the exercise training for 60 min per day for 6 days per week.</li>
		<li>For aftercare, refer to steps 2.1.4-2.1.5.</li>
		<li>After exercise, give the EX rat 5 g of rodent sunflower seeds to provide nutritional support.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>After each swim training session, clean and disinfect all equipment with ultraviolet light, including water buckets and towels.</li>
</ol>
3. Physical characteristics of fetuses
<ol>
	<li>On the morning of GD20.5, anesthetize the female rat with 3% isoflurane and euthanize it in a CO2 chamber.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

Exploring Maternal Exercise for the Well-Being of Rat Offspring

<ol>
	<li>Hanson, M. A., Gluckman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+developmental+conditioning+of+later+health+and+disease:+Physiology+or+pathophysiology.">Early developmental conditioning of later health and disease: Physiology or pathophysiology.</a> Physiol Rev. 94 (4), 1027-1076 (2014).</li><li>Calkins, K., Devaskar, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+origins+of+adult+disease.">Fetal origins of adult disease.</a> Curr Probl Pediatr Adolesc Health Care. 41 (6), 158-176 (2011).</li><li>Chavatte-Palmer, P., Tarrade, A., Rousseau-Ralliard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+before+and+during+pregnancy+and+offspring+health:+The+importance+of+animal+models+and+what+can+be+learned+from+them.">Diet before and during pregnancy and offspring health: The importance of animal models and what can be learned from them.</a> Int J Environ Res Public Health. 13 (6), 586 (2016).</li><li>Lapehn, S., Paquette, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placental+epigenome+as+a+molecular+link+between+prenatal+exposures+and+fetal+health+outcomes+through+the+dohad+hypothesis.">The placental epigenome as a molecular link between prenatal exposures and fetal health outcomes through the dohad hypothesis.</a> Curr Environ Health Rep. 9 (3), 490-501 (2022).</li><li>Shrestha, A., Prowak, M., Berlandi-Short, V. M., Garay, J., Ramalingam, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+obesity:+A+focus+on+maternal+interventions+to+improve+health+of+offspring.">Maternal obesity: A focus on maternal interventions to improve health of offspring.</a> Front Cardiovasc Med. 8, 696812 (2021).</li><li>Haire-Joshu, D., Tabak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+obesity+across+generations:+Evidence+for+early+life+intervention.">Preventing obesity across generations: Evidence for early life intervention.</a> Annu Rev Public Health. 37, 253-271 (2016).</li><li>Kusuyama, J., Alves-Wagner, A. B., Makarewicz, N. S., Goodyear, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+maternal+and+paternal+exercise+on+offspring+metabolism.">Effects of maternal and paternal exercise on offspring metabolism.</a> Nat Metab. 2 (9), 858-872 (2020).</li><li>Blaize, A. N., Pearson, K. J., Newcomer, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+maternal+exercise+during+pregnancy+on+offspring+chronic+disease+susceptibility.">Impact of maternal exercise during pregnancy on offspring chronic disease susceptibility.</a> Exerc Sport Sci Rev. 43 (4), 198-203 (2015).</li><li>Hinman, S. K., Smith, K. B., Quillen, D. M., Smith, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+in+pregnancy:+A+clinical+review.">Exercise in pregnancy: A clinical review.</a> Sports Health. 7 (6), 527-531 (2015).</li><li>Acog committee. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity+and+exercise+during+pregnancy+and+the+postpartum+period:+Acog+committee+opinion,+number+804.">Physical activity and exercise during pregnancy and the postpartum period: Acog committee opinion, number 804.</a> Obstet Gynecol. 135 (4), e178-e188 (2020).</li><li>Poole, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+animal+exercise+and+training+protocols+for+cardiovascular+studies.">Guidelines for animal exercise and training protocols for cardiovascular studies.</a> Am J Physiol Heart Circ Physiol. 318 (5), H1100-H1138 (2020).</li><li>Beleza, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-paced+free-running+wheel+mimics+high-intensity+interval+training+impact+on+rats'+functional,+physiological,+biochemical,+and+morphological+features.">Self-paced free-running wheel mimics high-intensity interval training impact on rats' functional, physiological, biochemical, and morphological features.</a> Front Physiol. 10, 593 (2019).</li><li>August, P. M., Rodrigues, K. D. S., Klein, C. P., Dos Santos, B. G., Matt&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+gestational+exercise+practice+and+litter+size+reduction+on+maternal+care.">Influence of gestational exercise practice and litter size reduction on maternal care.</a> Neurosci Lett. 741, 135454 (2021).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+exercise+reprograms+the+development+of+hypertension+progress+and+improves+vascular+health+in+shr+offspring.">Prenatal exercise reprograms the development of hypertension progress and improves vascular health in shr offspring.</a> Vascul Pharmacol. 139, 106885 (2021).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+during+pregnancy+enhances+vascular+function+via+epigenetic+repression+of+ca(v)1.2+channel+in+offspring+of+hypertensive+rats.">Exercise during pregnancy enhances vascular function via epigenetic repression of ca(v)1.2 channel in offspring of hypertensive rats.</a> Life Sci. 231, 116576 (2019).</li><li>Musial, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+alters+the+molecular+pathways+of+insulin+signaling+and+lipid+handling+in+maternal+tissues+of+obese+pregnant+mice.">Exercise alters the molecular pathways of insulin signaling and lipid handling in maternal tissues of obese pregnant mice.</a> Physiol Rep. 7 (16), e14202 (2019).</li><li>Dos Santos, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+exercise+was+safe+for+the+pregnancy+and+offspring's+development+and+partially+protected+rats+against+early+life+stress-induced+effects.">Resistance exercise was safe for the pregnancy and offspring's development and partially protected rats against early life stress-induced effects.</a> Behav Brain Res. 445, 114362 (2023).</li><li>Barnes, M. D., Heaton, T. L., Goates, M. C., Packer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersystem+implications+of+the+developmental+origins+of+health+and+disease:+Advancing+health+promotion+in+the+21st+century.">Intersystem implications of the developmental origins of health and disease: Advancing health promotion in the 21st century.</a> Healthcare. 4 (3), 45 (2016).</li><li>Bakrania, B. A., George, E. M., Granger, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+preeclampsia:+Investigating+pathophysiology+and+therapeutic+targets.">Animal models of preeclampsia: Investigating pathophysiology and therapeutic targets.</a> Am J Obstet Gynecol. 226, S973-S987 (2022).</li><li>Ferrari, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+during+pregnancy+and+its+impact+on+mothers+and+offspring+in+humans+and+mice.">Exercise during pregnancy and its impact on mothers and offspring in humans and mice.</a> J Dev Orig Health Dis. 9 (1), 63-76 (2018).</li><li>Lerman, L. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+hypertension:+A+scientific+statement+from+the+american+heart+association.">Animal models of hypertension: A scientific statement from the american heart association.</a> Hypertension. 73 (6), e87-e120 (2019).</li><li>Mangwiro, Y. T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+initiated+during+pregnancy+in+rats+born+growth+restricted+alters+placental+mtor+and+nutrient+transporter+expression.">Exercise initiated during pregnancy in rats born growth restricted alters placental mtor and nutrient transporter expression.</a> J Physiol. 597 (7), 1905-1918 (2019).</li><li>Kim, H., Lee, S. H., Kim, S. S., Yoo, J. H., Kim, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+maternal+treadmill+running+during+pregnancy+on+short-term+memory+and+hippocampal+cell+survival+in+rat+pups.">The influence of maternal treadmill running during pregnancy on short-term memory and hippocampal cell survival in rat pups.</a> Int J Dev Neurosci. 25 (4), 243-249 (2007).</li><li>Harris, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+3'-sialyllactose+in+breast+milk+is+a+critical+mediator+to+improve+metabolic+health+and+cardiac+function+in+mouse+offspring.">Exercise-induced 3'-sialyllactose in breast milk is a critical mediator to improve metabolic health and cardiac function in mouse offspring.</a> Nat Metab. 2 (8), 678-687 (2020).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+exercise+represses+nox4+via+sirt1+to+prevent+vascular+oxidative+stress+and+endothelial+dysfunction+in+shr+offspring.">Maternal exercise represses nox4 via sirt1 to prevent vascular oxidative stress and endothelial dysfunction in shr offspring.</a> Front Endocrinol. 14, 1219194 (2023).</li><li>Shan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+exercise+upregulates+the+DNA+methylation+of+agtr1a+to+enhance+vascular+function+in+offspring+of+hypertensive+rats.">Maternal exercise upregulates the DNA methylation of agtr1a to enhance vascular function in offspring of hypertensive rats.</a> Hypertens Res. 46 (3), 654-666 (2023).</li></ol>

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 factors15,16. 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 behavior13,17 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&#160;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 studies15,16,21,22,23,24 (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 &#177; 1&#176;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&#39;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 &#945;1C (Cacna1c) gene, angiotensin II type 1 receptor (Agtr1a) gene and NADPH oxidase-4 (Nox4) gene to improve vascular function in hypertensive offspring14,25,26.
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.

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 health5,6.
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&#39;s safety and profound benefits, including substantial reductions in common pregnancy conditions such as gestational diabetes mellitus9,10. 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 models13,14, 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.

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

swimming exercise protocol and care methods for pregnant rats

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.

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&#39; 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&#39;s vagina to prevent the male rat&#39;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&#160;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&#39;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&#160;rat&#39;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66577/66577fig01.jpg" /> 
Figure 1: Rat pregnancy model. (A) The vaginal smear results at different estrous periods, rats&#39; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66577/66577fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66577/66577fig02.jpg" /> 
Figure 2: The pregnant rats&#39; body weight and the fetuses&#39; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66577/66577fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66577/66577fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66577/66577fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66577/66577fig04v2.jpg" /> 
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 &#177; 1 &#176;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66577/66577fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Possible problems in swimming exercise and postpartum care for pregnant rats and their solutions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66577/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66577/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Read this Scientific Article Protocol about Author Spotlight: Enhancing Offspring Health with Maternal Exercise During Pregnancy at JoVE.com

Author Spotlight: Enhancing the Offspring Health in Rats with Maternal Exercise During Pregnancy

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.

Swimming Exercise Protocol and Care Methods for Pregnant Rats

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, ...

swimming-exercise-protocol-and-care-methods-for-pregnant-rats

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

634 Views.  Beijing Sport University. 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.

Video: Author Spotlight: Enhancing the Offspring Health in Rats with Maternal Exercise During Pregnancy

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.

Detailed instructions are provided on how to train rats to voluntarily dive underwater through a 5 m long Plexiglas maze. Because the brains of rats have been very well characterized, voluntarily diving rats may help elucidate the central pathways of the mammalian diving response.

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a ...

Behavior

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Medicine

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Developmental Enhancement in Cerebral Palsy Rats via Spinal Manipulations

Enhancing the Development and Growth of Infant Cerebral Palsy Rats Using Selective Spinal Manipulations

Cerebral palsy (CP) is a refractory pediatric disease with a high prevalence, high disability rate, and difficult treatment. A variety of treatments are currently used for CP. The treatment involves drug and non-drug therapy. Traditional Chinese medicine external therapy is a very distinctive treatment method in non-drug therapy. As one of the external therapies of traditional Chinese medicine, massage is used in treating cerebral palsy and has good efficacy, small side effects, and strong operability. As a part of TCM external therapy, selective spinal manipulation can effectively promote the growth and development of infant rats with cerebral palsy.The operation was mainly divided into four steps: first, the rubbing method was applied to the spine and both sides of the spine for 1 min. The pressing and kneading method was applied to the spine for 5 min, and the muscles on both sides of the spine for 5 min. Second, pressing and kneading the sensitive local acupoints in the spine for 2 min were performed. Thirdly, the affected limb was treated by twisting method for 1 min. Fourth, the rubbing method was applied to a midline from the forehead to the back of the brain for 1 min. This study aimed to use selective spinal manipulation to treat infant rats with cerebral palsy. The weight, Rotarod test, Foot-fault score, and growth hormone of infant rats with cerebral palsy were detected to understand the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy. The results showed that it can promote weight gain, improve balance ability and motor function, promote growth and development of infant cerebral palsy rats, promote growth hormone secretion, and increase the temperature of sensitive parts of the back.

This study illustrates the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy, emphasizing the specific procedure and standardized protocol. Body weight measurement, Rotarod test, Foot-fault score, other behavioral tests, and growth hormone detection were performed to evaluate the protocol.

Cerebral palsy (CP) is a refractory pediatric disease with a high prevalence, high disability rate, and difficult treatment. A variety of treatments are ...

Neuroscience

Creating a Preterm Rat Model for Early Postnatal Pain Research

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...

Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology. 
Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 ...