Modifying Levels of Maternal Dietary Folic Acid or Choline to Study the Impact of Deficiencies on Offspring Health Outcomes

Summary

This protocol describes the method for reducing dietary intake of folic acid or choline in female mice prior to pregnancy with the objective of investigating the impact of maternal diet on offspring health outcomes.

Abstract

Maternal nutrition during pregnancy and lactation plays an important role in the neurodevelopment of offspring. One-carbon (1C) metabolism, which centers around folic acid and choline, as well as other B vitamins, plays a key role during the closure of the neural tube of the developing fetus. However, the impact of these maternal nutritional deficiencies during pregnancy on offspring health outcomes after birth remains relatively undefined. Furthermore, maternal dietary deficiencies in folic acid or choline may impact other health outcomes in offspring - making this a valuable model. This protocol aims to outline the procedure for inducing a deficiency in 1C metabolism in female mice through dietary modifications. Females are placed on diets at weaning, up to 2 months of age, for 4-6 weeks prior to mating and remain on diet throughout pregnancy and lactation. Offspring from these females can be evaluated for health outcomes. Females can be used multiple times to generate offspring, and tissues from females can be collected to measure for 1C metabolite measurements. This protocol provides an overview of how to induce maternal dietary deficiencies in folic acid or choline to study offspring health outcomes.

Introduction

Maternal nutrition during pregnancy and lactation plays a vital role in offspring development. One-carbon (1C) metabolism is a key metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, and epigenetics. Folic acid and choline are central components of 1C that play an important role in the closure of the neural tube. It is widely recommended for women to supplement with folic acid and choline during their childbearing years¹. While the critical role of folic acid in fetal development is clearly established, the underlying mechanisms involved remain unclear. Women of childbearing are recommended to take folic ....

Protocol

This protocol was approved, and all the animal subjects were used in accordance with the guidelines of the Institutional Animal Care and Use Committee of Midwestern University (Protocol 2983). Female C57BL/6J mice, 2 months of age (body weight, ~20 g), were used in this study. In general, female mice of reproductive age can be used¹⁰. To ensure litter effects are avoided, multiple females need to be used for each diet¹¹. The details of the reagents and the equipment used in.......

Discussion

During the implementation of this protocol in research laboratories, there are several important notes that researchers need to consider prior to starting experiments. Firstly, in diet manufacturing, enough lead time needs to be given to manufacture the diet before the start of the experimental study. Lead times can vary from company to company, and shipment of diet also needs to be factored in. Female mice that are at reproductive age (~2-2.5 months in age) can be placed on the diet 4-6 weeks prior to mating

Materials

Name	Company	Catalog Number	Comments
1.5 mL tubes	Eppendorf
C57BL/6 Female Mice	Charles River	27	Mice can be bred in house as well.
Choline Deficient Diet	Envigo	TD.06119
Control Diet	Envigo	TD.190790
EDTA tubes
Folic Acid Deficient Diet	Envigo	TD.01546
GraphPad prism			https://www.graphpad.com/features
Housing for rodents	Lab Products, LLC	One CageVentilated Racks & Cages
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)	SCIEX, Framingham, MA	5500 QTrap
Ultra-performance liquid chromatography (UPLC)	Shimadzu, Columbia, MD, USA	Nexera