Physiological Effects of Isocaloric and Mineral Deficient Maternal Mouse Diets on Offspring Growth and Body Composition

PHYSIOLOGICAL EFFECTS OF ISOCALORIC AND MINERAL DEFICIENT MATERNAL MOUSE DIETS ON OFFSPRING GROWTH AND BODY COMPOSITION

A THESIS SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY

Amanda Palowski

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

CO-ADVISORS

Christopher Faulk, Gerald C. Shurson

August 2018

Ó Amanda Palowski 2018

Acknowledgements First, I would like to acknowledge and express my thankfulness to my academic advisors, Drs. Gerald Shurson and Christopher Faulk. Both of my advisors have challenged me to think outside of the box, pushed me to keep improving, and provided me with the knowledge and tools to be successful in the research field. I would not be the student and scientist that I am today without their guidance and support. Secondly I would like to thank all of the Integrated Animal Systems Biology professors. It has been a great pleasure working with all of you and I thank you for your guidance and help throughout my graduate career. I would also like to thank Dr. Dan Gallaher for being a part of my committee and providing an outside set of eyes on my project and thesis, I appreciate all of your comments and guidance. I would also like to thank all of the graduate and undergraduate students who helped me with my project; Mathia (Tia) Colwell, Mickie Trudeau, Yuan-tai Hung, Anna Clarke, Melissa Drown, and Chelsea Drown. I also want to thank all of the other graduate students in our department, Marta Ferrandis, Joey Mielke, Leonard Fung, Zhikai Zeng, Jinlong Zhu, Edward Yang, Audrey Walter, and Julia Holen, thank you for showing me friendship and helping me with classes. Lastly I would like to thank my family for the constant support along this journey to further my education, especially my husband Adam. It is never easy buying a house, getting married and starting a family in the middle of grad school, but he showed me constant love and support and I appreciate you pushing me to be a better person.

Dedication This thesis is dedicated to my husband Adam Palowski. Thank you so much for being my best friend and all of the love and support you have shown me through this journey. Thank you for always pushing me and being there to help me pick myself up and dust myself off when I stumble and fall. Thank you for being the best support system ever, I love you so much.

Abstract Over the last 30 years, researchers have evaluated the various effects of feeding maternal high fat diets on the health of the offspring of various species, but mainly in rodents. Our hypothesis was that dietary source and amount of energy in maternal mouse diets would induce physiological, growth, and body composition changes in offspring independently of maternal body weight. The objective of this study was to evaluate the changes in body weight and composition from feeding maternal isocaloric diets on mouse offspring (1 generation). Parent mice were weaned and fed one of 3 diets consisting of either low fat with controlled calories based on prior ad libitum mouse experiment measurements (LFr), high fat diets restricted in calories to match the low fat diet (HFr), or high fat ad libitum (HFa) diets. Offspring from the first generation were fed either low fat ad libitum (LFa) or high fat ad libitum (HFa) diets and randomly assigned to four different dietary treatment groups: 1) dams fed LFr and offspring fed LFa (LFr:LFa) diets, 2) dams fed LFr and offspring fed HFa (LFr:HFa) diets, 3) dams fed HFr and offspring fed HFa (HFr:HFa) diets, and 4) dams fed HFa and offspring fed HFa (HFa:HFa) diets. Average weekly body weight and composition of offspring was determined for each of the four dietary treatments. Unfortunately, the experimental diets obtained from the manufacturer were severely deficient in all of the essential macro and micro minerals, which resulted in high pup mortality and prevented testing of the original hypothesis. Overall, both dams and offspring fed the LF diet had the greatest body weight gain and average percentage of body fat mass compared with those fed the other dietary treatments. In conclusion, feeding the mineral deficient diets confounded the effects of dietary calorie source and consumption on developmental programming in mice.

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Table of Contents Acknowledgements i Dedication ii Abstract iii List of Figures v List of Tables viii Chapter 1: Literature Review 1 Introduction 1 Developmental Programming 1 Maternal Nutritional Effects of Developmental Programming 4 Maternal Malnutrition and Effects on Offspring 5 Molecular Mechanisms of Maternal Nutritional Effects 7 Effects of Maternal High Fat Diet on Offspring Metabolic Health 9 Maternal High Fat Diet Effects on Milk Production 14 Maternal High Fat Diet Effects on Offspring Hepatic Function and Metabolic Syndrome 15 Mental Health Effects of Maternal Diets 16 Comparison of Dietary Energy Systems 17 Effect of Mineral Deficiencies on Reproduction and Growth of Mice 22 Conclusions 27 Chapter 2: Physiological Effects of Isocaloric and Mineral Deficient Maternal Diets On Offspring Growth and Body Composition 29 Synopsis 29 Introduction 30 Materials and Methods 32 Results and Discussion 45 Conclusion 60 Overall Summary 61 References 63

List of Figures Figure 1. Partitioning of dietary energy losses and utilization (National Research Council, 2012)...…………………………………………………………….19 Figure 2. Experimental design of dietary treatments of parent (F0) mice……….33 Figure 3. Daily amount of food provided to female mice fed low fat restricted (LFr) and high fat restricted (HFr) diets compared with the amount of food (g) consumed by the female mice fed the high fat ad libitum (HFa) dietary treatment…...…………………………………………………….………….40 Figure 4. Daily amount of calories offered to female mice fed low fat restricted (LFr) and high fat restricted (HFr) diets compared with the amount of calories consumed by the female mice fed the high fat ad libitum (HFa) dietary treatment.………………..………………….………………………40 Figure 5. Comparison of the weekly calorie consumption by female mice fed the 2018 diet (Atwater calories), the high fat ad libitum diet (HFa; Atwater calories) and HFa (NE calories system calculations)…………………...41 Figure 6. Timeline of significant events that occurred throughout the study…….42 Figure 7. Experimental design of dietary treatments of F1.2 mice offspring (first diet abbreviation represents the dam diet and the second diet abbreviation represents the offspring diet)…………………………………………………43 Figure 8. Average weekly body weight (g) of the dams fed low fat restricted (LFr; n = 8), high fat restricted (HFr; n = 6), of high fat ad libitum (HFa; n = 8) diets………………….….....…………………………………………………...46 Figure 9. Average weekly body weight (g) of the male offspring among the four maternal and offspring dietary treatment combinations with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 9), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 13), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 19), and HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 12)…………………………………………………47 v

Figure 10. Average weekly body weight (g) of the female offspring among the four maternal and offspring dietary treatment combinations with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 7), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 10), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 9), and HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 16)…………………………………………………48 Figure 11. Average percentage of body fat (A) and fat-free mass (B) of dams at 24 weeks of age among the three maternal dietary treatments with LFr representing low fat restricted diet (n = 8), HFr representing high fat restricted diet (n = 6), HFa representing high fat ad libitum diet (n = 8). Different letters indicate statistically significant differences……………….50 Figure 12. Average percentage of body fat (A) and fat free mass (B) of offspring at 12 weeks of age among the four maternal and offspring dietary treatment combinations with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 16), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 23), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 28), and HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 28)…….51 Figure 13. Average percentage of fat composition between male and female offspring among the four maternal and offspring dietary treatment groups. LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 9 males and 7 females), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 13 males and 10 females), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 19 males and 9 females), HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 12 males and 16 females)….……………………………52

Figure 14. Average percentage of fat free mass composition between male and female offspring among the four maternal and offspring dietary treatment groups. LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 9 males and 7 females), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 13 males and 10 females), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 19 males and 9 females), HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 12 males and 16 females)………………………53 Figure 15. Histology of a liver section from a dam fed the high fat maternal diet stained with Oil Red O stain for fat content at 10x magnification………...55 Figure 16. Histology of a liver section from the offspring fed the maternal and offspring dietary treatment group HFa:HFa (A) representing the high fat ad libitum dam diet and high fat ad libitum offspring diet and HFr:HFa (B) representing high fat restricted maternal diet and high fat ad libitum offspring diet stained with Oil Red O stain for fat content at 10x magnification…………………………………………………………………...56 Figure 17. Histology of a liver section from the offspring fed the maternal and offspring dietary treatment group LFr:HFa (A) representing the low fat restricted dam diet and high fat ad libitum offspring diet and LFr:LFa (B) representing low fat restricted maternal diet and low fat ad libitum offspring diet stained with Oil Red O stain for fat content at 10x magnification..….57

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List of Tables Table 1. Summary of rodent maternal dietary lipid levels, feeding duration, and offspring responses*……………….…………………………………………10 Table 2. Calculated Atwater and NE content of the mouse AIN-93G diet a well- defined and standard mouse diet) based on chemical composition and digestibility estimates……………..……………...……………………………20 Table 3. Summary of macro- and micro- mineral elements required for mouse diets, major functions, and deficiency signs………………………………..22 Table 4. Calculated nutrient composition of the low fat and high fat experimental diets……………………………………………………………………………..34 Table 5. Ingredient composition (%) of the low fat and high fat experimental diets………………..……………………………………………………………36 Table 6. Comparison of the calculated energy content using the Atwater and net energy (NE) systems and chemical components in the low fat and high fat experimental diets..……………………………………………………………37 Table 7. Comparison of formulated and analyzed macronutrient composition of the low fat diet, high fat diet, and NRC (1995) requirements (mg/kg)……58 Table 8. Comparison of formulated and analyzed mineral composition of the low fat diet with NRC (1995) requirements (mg/kg)………………………….…59 Table 9. Comparison of formulated and analyzed mineral composition of the high fat diet with NRC (1995) requirements (mg/kg)…………………………….60

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Chapter 1: Literature Review Introduction In today’s society, over 70% of adults over the age of 20 in the United States are overweight or obese (Centers for Disease Control and Prevention, 2017). Although this statistic is disturbing, it is not surprising because of the widespread availability and consumption of foods that contain high amounts of fat (lipids) and simple sugars known as monosaccharides (e.g. glucose, fructose, galactose). In addition to consuming high calorie diets, many Americans do not engage in adequate exercise to maintain or reduce body weight gain resulting from high calorie intake. However, the inadequate balance between diet and exercise is not the only cause of obesity. Other factors such as maternal age, genetics, and most recently, epigenetics have been shown to contribute to obesity of humans (McAllister et al., 2009). Epigenetics is defined as the change in gene expression without changing the underlying DNA sequence (Berger et al., 2009). Evolution can provide a perspective and insights on dietary responses and other environmental factors that influence the prevalence of obesity. Obesity and other metabolic diseases, such as diabetes, are influenced by maternal environment (maternal age, maternal diet, maternal body weight). Multiple studies (Faulk et al., 2014; Huypens et al., 2016; Masuyama and Hiramatsu, 2012; Ravelli et al., 1976; Schulz, 2010; Smith, 1976) have shown that the early life fetal environment influences adult responses via developmental programming including dietary composition. Developmental programming Adult obesity and other physiological effects can occur from individual exposure to various environmental factors, but are also influenced by the previous maternal exposure to environmental factors during conception and fetal development. Animals are very capable of adapting to an ever-changing environment, and in some cases, can prepare for drastic environmental changes such as famine. There are several theories that relate to how such occurrences may occur. 1

In 1962, James Neel developed the ”thrifty gene hypothesis” in an attempt to understand the apparent high heritability of harmful metabolic disorders, such as Type II diabetes (Genné-Bacon, 2014; Neel, 1962). Neel (1962) proposed that gene variants that were once advantageous for survival, are now harmful. He reasoned that in pre-historic periods, this phenomenon would be very beneficial for hunter-gatherers because women would gain body weight and fat mass quickly in time of plentiful food, which increased their chances of survival during times of limited food intake or famine. Today, human famine is rare, and is certainly not a feature of the western lifestyle. Therefore, this trait has become very maladaptive and harmful. Neel (1962) also hypothesized that 2 to 3 generations of study would be required to determine if the “thrifty gene hypothesis” was true. However, there is a conspicuous lack of evidence to support this hypothesis, even though Neel himself (1962) attempted to find additional evidence that “thrifty” genes store energy in the body to prepare for a time of famine. In 1990, David Barker used epidemiological evidence to show that intrauterine growth retardation, low birth weight, and premature birth have a causal relationship to hypertension, coronary heart disease, and non-insulin dependent diabetes in adults (Genné-Bacon, 2014). This hypothesis was described as the “thrifty phenotype hypothesis”, which was a modification of the “thrifty gene hypothesis” proposed by Neel (1962). Barker (1990) created the description of “fetal origins of adult disease”, which has also been described as developmental origins of adult disease (DoHAD; Vaag et al., 2012). As shown from Barker (1990) epidemiological data, early life environment exposure corresponded to the propensity for developing adult metabolic diseases. In 2008, Richard Stoger combined the “thrifty genotype hypothesis” with the “thrifty phenotype hypothesis”, and suggested that every human has some inherent level of “thriftiness” due to an ancient, inherited system for conserving body stores of energy and nutrients, and that an individual’s disease risk is determined by epigenetic events, known as the “thrifty epigenome hypothesis” (Genné-

Bacon, 2014). From this hypothesis, the phrase “developmental programming” was introduced. Baig et al. (2011) developed a theoretical model that suggested that “thrifty” genes and fetal programming do not adequately account for substantial trends in obesity epidemics. In contrast to the work by Baig et al. (2011), Masuyama and Hiramatsu (2012) conducted a study that showed a metabolic syndrome-like phenomenon in offspring that may have been caused by consuming a maternal high lipid diet, resulting in epigenetic modifications to the expression of certain genes, giving a mechanistic underpinning to developmental programming. Developmental programming was initially supported by the Dutch Hunger Winter study, and later confirmed by subsequent additional studies (Ravelli et al., 1976; Smith, 1976). The Dutch Hunger Winter study refers to an episode during World War II, when trade into the Netherlands was blocked by the Nazis. In response to this blockade, the Dutch government limited the food rations of the citizens to 400 to 800 calories per person per day. It was assumed that the citizens were restricted uniformly in both calories and macronutrients as well as micronutrients (vitamins and minerals). After the war, researchers evaluated the health of children from mothers who were pregnant during this time to determine if there were any lasting effects of this caloric and nutritional restriction. By the 1970’s, these children that had become adults and had greater body mass index (BMI), increased cardiovascular disease, depression, and mental health disorders than their siblings born to the same mothers with normal caloric intake (consuming a minimum of 1,800 calories) during pregnancy. These adverse health conditions coincided with notable changes in DNA methylation in growth and metabolism genes (Ravelli et al., 1976; Smith, 1976). The Dutch Hunger Winter study also showed the importance of the timing of famine exposure in relation to developmental programming. Pregnant women who were subjected to low calorie intake during late gestation had lower rates of obesity as adults, but their offspring had low body weight at birth, as well as lower body weight and obesity rates throughout their lives compared with offspring born before or after famine

(Schulz, 2010). However, pregnant women who were exposed to caloric restriction during early gestation had increased rates of adult obesity, while women exposed low calorie intake during mid-pregnancy had indicators of reduced renal function (Schulz, 2010). It is unknown whether the effects of these adults were a result of only a caloric deficiency, or a combination of a caloric and micronutrient deficiency. Furthermore, the effects of maternal body composition on fetal development is unknown. Maternal Nutritional Effects of Developmental Programming A maternal effect is commonly defined as the effect of the maternal environment on the phenotype of offspring (Jiménez-Chillarón et al., 2014), which includes the direct contribution of the energy and nutrient intake of the mother, as well as oxygen supply and mRNA to the oocyte or fetus prior to birth. The contributions of the mother, especially nutrition, rather than the genotype of the offspring, influence a variety of factors in offspring including body weight. Due to the controversy over the accuracy of the “thrifty genotype” hypothesis, Huypens et al. (2016) conducted a study to determine if the susceptibility of offspring to obesity and Type 2 diabetes was increased by epigenetic inheritance alone (i.e. via gametes), thereby limiting the influence of maternal effect factors. These researchers fed either a high lipid diet with 60% of metabolizable energy coming from fat, a low lipid diet with 11% of metabolizable energy coming from fat, or a normal diet with 13.5% of metabolizable energy coming from fat, to male and female mice for 6 weeks. They evaluated 5 dietary treatment combinations consisting of 1) low lipid male diet x low lipid female diet, 2) normal chow male diet x normal chow female diet, 3) high lipid male diet x high lipid female diet, 4) high fat male diet x normal chow female diet, and 5) normal chow male diet x high fat male diet. Using in vitro fertilization, embryos were transplanted into foster does to determine if gametes were responsible for transferring the phenotype responses by removing confounding effects of gametes from the “maternal effect” of the gestational environment in developmental programming. At weaning, all offspring were fed a high lipid diet

and were observed for expression of characteristics of obesity and Type 2 diabetes. Results from this study showed that offspring from both parents fed the high lipid diet before mating, and offspring from only one parent fed the high lipid diet, had greater body weight and fat mass than offspring from parents fed the low lipid x low lipid and normal diet x normal diet treatments. These results indicate that there is equal propensity for obesity in offspring from high fat fed paternal and maternal gametes, and gametes are necessary and sufficient for transferring epigenetic information related to obesity and Type 2 diabetes. However, more research is needed to assess these effects in multiple subsequent generations. Similarly, Holland et al. (2016) conducted a study to determine the developmental programming epigenetic mechanisms of maternal protein restriction and found that offspring from protein restricted (8% protein) mothers weighed less at weaning, had reduced locomotor activity and glucose-stimulated insulin secretion, and hypermethylation of Rn45s on chromosome 17 compared with mice fed control diets (20% protein; Holland et al., 2016). Results from this study showed that early life nutrition can affect the genome and methylation of genes of mice. The overall results from these studies suggest that both maternal and paternal genders can influence on the genome and metabolic health of the offspring. However, the studies by Huypens et al. (2016) and Holland et al. (2016) did not adequately determine the role of parental diet exposure compared with parental phenotype on the developmental programming of offspring. While information related to parental diet can be transferred by gametes alone, it remains unclear whether developmental programming is truly from the diet or a combination of diet and parental physiology (e.g. weight or fat mass). Maternal Malnutrition Effects on Offspring Vonnahme et al. (2003) evaluated the effects of malnutrition of ewes in early- to mid-gestation on growth retardation of the offspring. Their hypothesis was that a 50% restriction of total digestible nutrients based on NRC (National Research Council, 1985) requirements, would result in a decrease in maternal

body weight gain and a reduction in the growth of the fetuses. These researchers used a 50% nutrient restriction because previous studies focused on moderate or decreasing nutrient restriction during gestation, but none of these studies evaluated the impact on placentomal vascularity. Placentomal vascularity is crucial for achieving a successful gestation in sheep, and fetal placental vascular development is comparable to that in humans. Ewes were fed either a control gestation diet, or a diet representing 50% of the recommended NRC (1985) energy and nutrient requirements beginning at 28 days of gestation. On day 78 of gestation, ewes were euthanized in order to evaluate the effects of early- to mid- gestation nutrition on fetal body and organ weights, fetal length, maternal and fetal blood, and umbilical cord blood. Ewes fed the energy and nutrient restricted diet had less maternal and fetal body weight than the ewes fed the nutritionally adequate diet. Furthermore, ewes fed the nutrient restricted diet had reduced blood glucose concentrations and increased T4 to T3 ratios, which indicates that T3 must be produced by peripheral tissues, and that the maternal blood glucose may be aiding in the metabolic function of the liver in the offspring. Fetuses from ewes in the nutrient-restricted group weighed less than the fetuses from the control ewes, yet the individual organ weights of the nutrient-restricted fetuses were not different between treatments (Vonnahme et al., 2003). These results suggest that nutrient restriction in early to mid-gestation may be harmful to the fetus postnatally, and potentially result in metabolic and cardiovascular disease later in life. Furthermore, these results mirror results observed in children from the Dutch Hunger Winter study. A variety of studies have determined that lifespan, and several important physiological effects such as cardiac health, immunity, insulin and glucose levels, can be increased when animals are fed calorically restricted and low-protein high-carbohydrate diets provided ad libitum (Lee et al., 2008; Masoro, 2005; Mattison et al., 2012; McCay et al., 1935; Mercken et al., 2012; Piper et al., 2011; Solon-Biet et al., 2015; Weindruch et al., 1986). However, these types of diets are not very applicable to humans because many people cannot maintain a

calorically restricted diet. Recognizing this issue, Solon-Biet et al., (2015) provided mice with ad libitum access to one of six diets: 1) high-protein, low- carbohydrate, 2) medium-protein, medium-carbohydrate, 3) low-protein, high- carbohydrate diets, and also fed calorically restricted diets consisting of 4) high- protein, low-carbohydrate, 5) medium-protein, medium-carbohydrate, and 6) low-protein, high-carbohydrate diets for 8 weeks. After 8 weeks of feeding these diets, mice that were fed the ad libitum low-protein, high-carbohydrate diet displayed similar metabolic improvements including improved homeostatic model assessment (HOMA), blood insulin and triglyceride concentrations, compared with mice that were fed the calorically restricted diets. Mice fed the ad libitum high-protein, low-carbohydrate diet had impaired glucose tolerance, as well as greater blood insulin and insulin resistance HOMA scores, compared with the other dietary treatment groups. However, mice provided ad libitum access to the low-protein, high-carbohydrate diet had improved blood insulin, HOMA scores, and triglyceride concentrations compared with mice provided ad libitum access to the other diets, and had similar concentrations of these measures with mice fed the calorically restricted diets. They also observed an increase in energy intake in the ad libitum low-protein, high-carbohydrate diet compared with feeding the ad libitum high-protein, low-carbohydrate diet, but there was no increase in fatty liver and body adiposity (Solon-Biet et al., 2015). Overall, the results from this study indicate that replacing a calorically restricted diet with an ad libitum low-protein high-carbohydrate diet provides the same health benefits without increasing fatty liver or adiposity accumulation. Molecular Mechanisms of Maternal Nutritional Effects Understanding the mechanism of maternal nutrition effects on subsequent generations of offspring physiology and development requires determining the genetic signals that are transferred from fetus to adulthood which are mediated by dam’s environment (Laubach et al., 2018). These epigenetic signals can be in the form of small RNA fragments, chromatin modification, or DNA methylation (Laubach et al., 2018).

Transgenerational inheritance patterns can be impacted by cytosine methylation, which is a very well characterized epigenetic marker (Laubach et al., 2018). Therefore, Shea et al. (2015) examined cytosine methylation patterns in mouse sperm in response to three post-weaning diets: 1) control (19% protein), 2) low-protein (10% protein), and 3) high fat (60% fat calories; Shea et al., 2015). After feeding these diets for 7 to 11 weeks, no effects from feeding the low- protein or high fat diets were observed on the methylation or copy number of loci. Therefore, these researchers concluded that the effects of diet composition had less effect on sperm methylome than stochastic epigenetic variation (e.g. environmental factors), and dietary composition did not play a role in sperm cytosine methylation. While paternal diets can influence the metabolism and metabolic health of offspring, evaluating these epigenetic changes via cytosine methylation may not be the most successful approach due to paternal diets not altering the cytosine methylation in paternal sperm. In contrast, evidence that maternal diet energy and nutrient restriction can act at the molecular level was reported by Cannon et al. (2014). They evaluated DNA methylation and genome-wide gene expression in liver, pancreas, white adipose, brain, muscle, and heart, along with body weight changes in mouse offspring from mothers fed a high lipid or low lipid diet. Results from this study showed that the changes in body weight of the offspring were associated with widespread gene expression due to maternal diet. Specifically, there was a well- defined effect on genes for RXR activation, inflammation, and cholesterol synthesis in the liver (Cannon et al., 2014). Molecular changes in the hippocampus, and a decrease in global methylation of the brain was observed when feeding maternal mouse diets providing 60% of calories from fat for at least 3 weeks prior to mating (Peleg-Raibstein et al., 2012; Vucetic et al., 2015). This means that maternal nutrition may cause long-term manipulations in gene expression, particularly in genes involved in inflammation and cholesterol synthesis.

A more recent study was conducted to determine if epigenetic inheritance or serial programming determined maternal programming (Eaton et al., 2017). Epigenetic inheritance is defined as the inheritance of an induced phenotype due to multi-generational effects. Serial programming is defined as the proliferation of the phenotype by the means of recurrent exposure to a compromised environment, particularly during gestation. In this study, weaned mouse offspring from obese dams were fed either a control, high fat, or high sugar Western-style diet. These researchers determined that male offspring in the first, second, and third generations had a predisposition to hepatic insulin resistance, but this was not observed unless the offspring were placed on the high sugar Western diet. These results exclude serial programming because the offspring were not continuously exposed to the compromised environment, but metabolic changes still occurred, suggesting that epigenetics results in maternal programming (Eaton et al., 2017). This means that feeding a high fat maternal diet predisposes offspring to obesity and Type 2 diabetes due to epigenetic inheritance, or the inheritance of a phenotype due to multi-generational effects instead of the recurrent exposure to a compromised environment (serial programming). Thus, traits like a predisposition to obesity and Type 2 diabetes can be passed through generations with just one exposure to a compromised in utero environment. This suggests that each dam does not need to continuously consume a high fat diet to influence offspring for subsequent generations. Therefore, maternal nutrition can continue to influence offspring through multiple generations. Effects of Maternal High Fat Diet on Metabolic Health of Offspring Numerous studies have evaluated the various effects of feeding a maternal high fat diet on offspring metabolic health (Table 1). However, although all of these studies evaluated feeding “high fat” diets, the dietary sources, concentrations, and time of feeding varied among studies. Three studies evaluated the effects of feeding a maternal high fat diet during gestation and lactation, while 17 studies determined the effects of feeding maternal high fat diets prior to mating through gestation and lactation (Table 1).

Table 1. Summary of rodent maternal dietary lipid levels, feeding duration, and offspring responses1. Reference Species Diet Composition Feeding Offspring Responses Duration Diets Fed During Gestation and Lactation Only Strakovsky et Rat 45% of calories Gestation PCK1 gene altered al. (2011) from fat (lard) (20 days histone modification, total) increased glucose and birth weight Gregorio et Mouse 49% of calories Gestation Insulin resistance, al. (2010); from fat (lard) and increase in hepatic Volpato et al. lactation (42 lipogenic genes, hepatic (2012) days total) triglycerides and hepatic steatosis Diets fed Prior to Mating Through Gestation and Lactation Elahi et al. Mouse 18% of calories 6 weeks Offspring predisposed to (2009) from fat (animal prior to metabolic syndrome, lard) mating, cardiovascular effects gestation may be sex-specific and lactation (84 days total) Lanham et al. Mouse 18% of calories 7 weeks Increase in adiposity of (2010) from fat (animal prior to bone marrow, altered lard) mating, bone structure gestation and lactation (91 days total)

Chechi et al. Mouse 20% fat from 2 weeks Reduction in hepatic (2010); polyunsaturated prior to and heart DHA content, Chechi and fatty acids or mating, SFA increased total Cheema saturated fatty gestation plasma and LDL (2006) acids (lard or and cholesterol safflower oil) lactation (56 days total) Hoile et al. Rat 21% of fat from 2 weeks Changes in vascular (2013); butter, fish oil, prior to function and the CpG Kelsall et al. trans fatty acids mating, methylation of specific (2012) gestation genes (FADS2 and promoter) lactation (56 days total) Franco et al. Rat 28% of calories 8 week prior Increased thyroid and (2012) from fat (lard) to mating, adrenal medullary gestation function, hyper and adiposity, lactation (98 hyperleptinanemia, days total) hyperglycemia Jones et al. Mouse 32% of calories 8 weeks Increase in fetal weight (2009) from fat (source prior to and placental GLUT1 not specified) mating, and SNAT2 mRNA gestation and lactation (98 days total) Ashino et al. Mouse 35% of calories 1 week prior Offspring with metabolic (2012) from fat (source to mating, syndrome, hepatic lipid not specified) gestation accumulation and and increased hepatic JNK lactation (49 phosphorylation

days total) Hartil et al. Mouse 36% of calories 2 weeks Decrease in fetal growth (2009) from fat (lard) prior to and birth weight and mating, increase in expression gestation of hepatic lipogenic and genes lactation (56 days total) Bruce et al. Mouse 45% of calories 4 weeks Reduced mitochondrial (2009) from fat (source prior to function, increased not specified) mating, hepatic lipogenesis, gestation oxidative stress, and and changes the expression lactation (70 of inflammatory genes days total) Dunn and Mouse 45% of calories 4 weeks Reduced insulin Bale (2009); from fat (lard and prior to sensitivity, altered Dunn and soybean oil) mating, growth hormone axis, Bale (2011) gestation increased body length, and altered expression of lactation (70 paternally imprinted days total) genes in F3 Zhang et al. Mouse 52% of calories 6 weeks Increased birth weight, (2009, 2005) from fat (source prior to increased protein levels not specified) mating, in fatty acid oxidation, gestation altered hepatic and microRNA lactation (84 days total) Gniuli et al. Mouse 60% of calories 8 weeks F1 and F2 generations (2008) from fat (saturated prior to had Type 2 Diabetes, fatty acids; other mating, higher beta cell mass in

sources not gestation F1, lower beta cell mass specified) and in F2 lactation (98 days total) Krasnow et Mouse 60% of calories 4,12, or 23 No effect on hepatic al. (2011) from fat (source weeks prior inflammation or not specified) to mating, expression of genes gestation involved in lipid and metabolism lactation (70, 126, or 203 days total) Peleg- Mouse 60% of calories 3 weeks Increased anxiety Raibstein et from fat (source prior to behavior, molecular al. (2012) not specified) mating, changes in the gestation hippocampus and lactation (63 days total) Vucetic et al. Mouse 60% of calories 12 weeks Decrease in global (2010) from fat (source prior to methylation in the brain not specified) mating, gestation and lactation (126 days total) 1 Definitions of abbreviations: PCK1 = phophoenolpyruvate carboxykinase 1; DHA = docosahexaenoic acid; SFA = saturated fatty acid; LDL = low density lipoprotein; CpG = cytosine nucleotide followed by guanine nucleotide; FADS2 = fatty acid desaturase 2; GLUT1 = glucose transporter 1; SNAT2 = sodium-

dependent neutral amino acid transporter 2; JNK = c-Jun N-terminal kinase; F1 = first generation; F2 = second generation; F3 = third generation. Results from studies summarized in Table 1 show that maternal diets have a significant influence on the physiology, epigenetics, and metabolism of offspring. In addition, although the maternal diet affects offspring, the composition of diets consumed by offspring is also important. Of the studies summarized in Table 1, five studies (Bruce et al., 2009; Chechi and Cheema, 2006; Elahi et al., 2009; Gniuli et al., 2008; Volpato et al., 2012) weaned offspring into 2 groups and either fed them the same diet as the dam’s diet, or switched to the opposite diet. Four studies (Ashino et al., 2012; Dunn and Bale, 2011, 2009; Vucetic et al., 2010) fed weaned offspring a control diet, while 3 studies (Hoile et al., 2013; Kelsall et al., 2012; Lanham et al., 2010) fed weaned offspring a high fat diet, and 4 studies (Chechi et al., 2010; Gregorio et al., 2010; Zhang et al., 2009, 2005) fed weaned offspring the same diet that their dam consumed. About 60% of the studies in Table 1 reported increases in cholesterol, hepatic function, genes associated with hepatic function, and presence of metabolic syndrome regardless of the composition of the diet fed to weaned offspring. Overall, the results from the majority of these studies support the concept that feeding a maternal high fat diet, regardless lipid source or feeding duration, predisposes offspring to metabolic syndromes or negatively impacts the hepatic function, as well as the genes involved in hepatic function of the offspring. However, there have been some inconsistent results from these maternal high fat studies in regard to fetal weight and birth weight responses. A few of studies reported an increase in fetal and birth weight in offspring from dams fed a high fat diet (Jones et al., 2009; Zhang et al., 2009, 2005), while other researchers reported a decrease in fetal and birth weight, which may be due to dietary lipid source, decreased maternal body weight gain, or food intake during gestation (Hartil et al., 2009). Because of these inconsistent responses, further research is needed to improve our understanding of feeding maternal high fat diets on fetal and birth weight responses of offspring. The studies by Hartil et al.

(2009), Jones et al. (2009), and Zhang et al. (2009, 2005) all fed potentially different lipid sources, different proportions of total dietary calories from lipids, and diets were fed for different time periods. Therefore, we cannot accurately determine the effects of feeding a maternal high lipid diet affects fetal and birth weight. Maternal High Fat Diet Effects on Milk Production The majority of the maternal high fat diet studies have focused on the effects of the diet on the offspring without being overly concerned with the effects on maternal health and well-being. One study examined the impacts of reduced calorie intake on milk production and metabolic events of the dams (Mohammad et al., 2009). These researchers compared feeding a reduced calorie, high carbohydrate diet with feeding an isonitrogenous, isocaloric high fat diet, and expected to see a reduction in milk production without impacting the weight of the dam in a short period of time. Although the short period of macronutrient deficiency had no effect on the volume of milk produced or the amount of protein and lactose in the milk, milk fat concentration was increased in dams fed the high fat diet compared to those fed the reduced calorie, high carbohydrate diet. As a result, the increased milk fat concentration increased the caloric content of the milk and consumption by the offspring even though milk production and milk energy output for both dietary treatment groups was comparable to published values of properly nourished lactating dams (Mohammad et al., 2009). These researchers concluded that a slight calorie restriction does not affect milk production, but feeding a maternal high fat diet increases milk fat and energy content resulting in a greater energy intake of offspring (Mohammad et al., 2009). The findings of this study support the concept that dams provide advantages to offspring in the form of higher energy content in milk when consuming a high fat diet. Implications of these findings suggest that dams consuming a high fat diet will offset any previous maternal caloric shortage by providing greater energy content in milk resulting in greater energy intake by the offspring, but additional research needs to be conducted to determine if a hypocaloric high fat diet would

result in greater maternal weight loss during lactation compared with feeding a high carbohydrate diet. Maternal High Fat Diet Effects on Offspring Hepatic Function and Metabolic Syndrome Multiple studies have shown a variety of responses from feeding maternal high fat diets prior to mating on hepatic function and metabolic syndrome, especially for lipogenesis and lipid accumulation in offspring (Ashino et al., 2012; Bruce et al., 2009; Dunn and Bale, 2011, 2009). As a side-effect of feeding dams high-fat diets, maternal obesity is induced and may directly affect the programming of the offspring. In addition, the effects of maternal diet have been shown to be additive or transmissible trans-generationally (Gniuli et al., 2008). When a diet providing 60% of the total calories from fat was fed prior to mating and throughout gestation and lactation, no effects were observed on hepatic inflammation or any expression of genes that are involved in lipid metabolism in offspring, but F1 generation offspring that had type 2 diabetes had greater beta cell mass while F2 generation offspring had lower beta cell mass (Gniuli et al., 2008; Krasnow et al., 2011). Adipogenesis in F1 mice offspring was inhibited, and osteoblastogenesis was enhanced, when dams were pair-fed an isocaloric high carbohydrate diet compared with dams pair-fed an isocaloric high fat diet (Mousavi et al., 2016). Guo and Jen (1995) found that offspring from dams fed a high fat diet weighed more and had greater body fat, as well as high blood glucose and triglyceride concentrations, which indicates that offspring may be predisposed to fatty liver and obesity. In addition, the retention of various effects from feeding a paternal high fat diet, including increased body weight gain and adipose tissue weights, has been observed in an controlled environment during pregnancy and the suckling period of offspring (Wu et al., 1998). The results of these studies, while somewhat contradictory, suggest that dams have an inherent greater priority for partitioning nutrients toward offspring than for maternal purposes. Mental Health Effects of Maternal Diets

Recent studies have reported that maternal obesity and/or maternal consumption of high fat diets can be risk factors for the mental health of offspring. An increase in offspring anxiety behavior was observed when feeding maternal mouse diets providing 60% of calories from fat for at least 3 weeks prior to mating (Peleg-Raibstein et al., 2012; Vucetic et al., 2015). Disruption of the mother-offspring relationship in rats has been shown to be associated with anxiety and early-life stress (altered hypothalamic-pituitary-adrenal axis response to stress, reduced hippocampal neurogenesis, altered emotionality, increased visceral pain and cognitive impairments; Aisa et al., 2007; Barreau et al., 2007; Francis et al., 2002; Kosten et al., 2012; Maniam and Morris, 2010; Nemeroff, 2016; Sánchez et al., 2001). However, feeding a maternal high fat diet can positively affect brain function of the offspring by relieving early life stressors (e.g. altered hippocampal neurogenesis, spatial learning deficits, and hyperanxiety; Rincel et al., 2016). Rincel et al. (2016) evaluated whether feeding a maternal high fat diet to rats would mimic maternal separation and the developmental alterations observed with maternal separation, and the long-term impact on maternal separation-induced alterations of emotional and cognitive behaviors. Their results showed that offspring behavior in adulthood was slightly positively affected by feeding the high fat maternal diet. A high fat maternal diet produced a protective effect on the molecular changes in brain development due to maternal separation. The maternal diet also reduced maternal anxiety in stressed dams. Maternal separation-induced endophenotypes, including anxiety, social behavior, stress response, visceral pain, and spatial memory in offspring were also reduced in response to maternal diet. Results from this study suggest that maternal obesity is a critical factor for brain development of the offspring, which is vulnerable to detrimental effects during development rather than dietary fat consumption, and that feeding a maternal high fat diet provides a protective effect during early-life stress (Rincel et al., 2016). Overall, even though there is inconsistent evidence showing that anxiety behavior is positively affected by feeding a maternal high fat diet, the results from these studies consistently show

that feeding maternal high fat diets increase the mental health of the offspring. Because many studies presume that feeding a maternal high-fat diet is the underlying cause of offspring dysfunction, without separating the confounding effect of induced maternal obesity, carefully designed studies are needed to better understand the role of diet and maternal obesity on mental health of offspring. Comparison of Dietary Energy Systems All of the diets in the studies previously reported, were formulated using metabolizable energy based on Atwater values, which is common method used when formulating rodent diets (Bielohuby et al., 2010). However, this is an inaccurate approach for formulating diets because it does not account for the digestibility and subsequent utilization of energy and nutrients of the ingredients and diets of rodents. Formulating diets for food producing animals involves using more advanced and accurate measures of energy and digestible nutrients to meet nutritional requirements. Therefore, interpreting and comparing nutritional responses among mouse studies is difficult because of dramatic differences in energy values of dietary ingredients used, and their inclusion rates. The Atwater system was developed by Wilber Atwater and is an FDA- approved method used to calculate the available energy in food (Oxford, 2006). Available energy is also known as metabolizable energy or the amount of gross energy that is available for use in the animal after accounting for energy losses from feces, urine, secretions, and gas production (Hill Laboratories, 2017). The Atwater system was determined experimentally by measuring heat from combustion of proteins, lipids, and carbohydrates. The resulting data provide caloric” factors” for protein, lipids, and carbohydrates that contribute variable amounts of calories in food. The caloric value of these chemical components is calculated by multiplying the dietary content (grams) by 4 (proteins), by 9 (lipids), and 4 (carbohydrates; Maynard, 1944). In 1991, Livesey discussed the seriousness of the errors in food energy values that are derived from imprecise calorie conversion factors (Atwater factors) and differences in food composition

(Livesey, 1991). In general, the use of Atwater factors usually leads to the overestimation of the energy value of a food ingredient or diet due to an increase in the amount of indigestible carbohydrate (fiber) content. It is difficult to compare responses among diets with different carbohydrate composition because of the variation in the true utilizable energy that each carbohydrate provides to the animal. Therefore, there is a tremendous need for using energy systems that accurately represents the proportion of gross energy (total calories) that are digested and fermented in the animal’s gastrointestinal tract and contribute to actual energy available. This is necessary for animal nutritionists, dieticians, and consumers to accurately characterize the utilizable energy value of various carbohydrate sources, especially for food-labelling claims (Livesey, 1991). The majority of human, rodent, laboratory, and companion animal studies ignore the energy losses in secretions and gases when formulating diets, and only consider losses from feces and urine. Therefore, it is important to understand differences among energy systems, deficiencies in using the Atwater system, and the need to begin using the net energy system to improve energy formulations in rodent diets. For food producing animals, feed energy is measured as gross energy (GE), digestible energy (DE), metabolizable energy (ME) and net energy (NE). According to the swine Nutritional Research Council (NRC, 2012), GE is defined as “the amount of energy produced when a compound is completely oxidized”, and DE is defined as “the result of subtracting the GE in the feces from dietary GE”. The swine NRC (2012) also defines metabolizable energy as “digestible energy minus the GE in urine” (National Research Council, 2012). As shown in Figure 1, the determination of NE accounts for losses in energy from heat increment and fermentation gases in addition to losses of energy from feces and urine (Whitney, 2017).

Feces Gas/Urine NEmaintenance GE DE ME

NEgrowth, gestation, lactation Heat

Figure 1. Partitioning of dietary energy losses and utilization(National Research Council, 2012).

While there are modifications to the Atwater system, it remains inaccurate for various dietary components and compositions (A.R.C Food Research Institute, 1981). Since the NE system accounts for heat increment, fecal, urinary, and gas losses, it is a more accurate system to use for estimating ingredient and dietary energy values than the Atwater system. This is important because heat losses vary among animal species, age, and size. In animal production, optimizing caloric and nutritional efficiency of diets is critical for minimizing feed cost, which represents 60 to 70% of the total cost of production. Therefore, use of the NE system is essential for accounting for the actual utilizable energy in feed ingredients, especially those with high fiber content, in precision animal nutrition programs. The digestible and utilized energy content in nuts and low-fat, high-fiber foods are overestimated using the Atwater system (Zou et al., 2007). Human patients are recommended to consume a high-fiber diet in order to reduce food intake, yet when this happens the energy availability as measured by the Atwater system is overestimated (Brown et al., 1998; Zou et al., 2007). Zou et al. (2007) observed an 11% difference between calculated Atwater factor values and determined ME values for low-fat, high-fiber human diets, which indicates that Atwater values for these types of foods are inaccurate and overestimate the energy availability. Consumption of low-fat, high-fiber diets often results in decreased food intake, which further would reduce daily energy intake (Zou et al., 2007). Similar findings have been reported by Novotny et al. (2012), who 20

determined the actual ME values of almonds and reported that using the Atwater factors overestimated the energy content by 32%. The NE system is beneficial in both human and animal diets that are high in fiber because of significant energy losses from heat produced during fermentation in the lower gastrointestinal tract, which is accounted for when using the NE system. Table 2 shows the comparative differences between the Atwater calculated energy values ((4 Kcal/g * protein) + (9 Kcal/g * fat) + (4 Kcal/g * carbohydrate)) and the NE calculated values (NE (MJ/kg) = (11.3*digestible crude protein + 35*digestible crude fat + 14.4*digestible crude fiber + 12.1 *digestible nitrogen-free extractives – 0.63*sugars)/1000) in a commonly used laboratory mouse diet, AIN-93G. By using the NE system, it is assumed that the net energy content from the food energy will be used for productive purposes as well as maintenance. However, total caloric intake is also dependent on food intake. These values show that the Atwater system does in fact underestimate the energy (% of calories) of protein, digestible carbohydrates and total nutrients. The Atwater factor also does not take into account fermentable carbohydrates, which underestimates the true energy value of carbohydrates. It is important to account for fermentable carbohydrates because they produce volatile fatty acids that can be absorbed and ultimately contribute to energy.

Table 2. Calculated Atwater and net energy (NE) content of the mouse AIN-93G diet (a well-defined and standard mouse diet) based on chemical composition and digestibility estimates. Nutrients Atwater energy Proportion of NE content content from various from various chemical chemical components, components, % of total % of total NE calories NE calories Crude protein 18.6 21.9 Digestible carbohydrates 65.0 76.4 Fermentable carbohydrates - 3.5

Lipids 16.4 13.5 Total 100 115.2

The total energy content for the Atwater system is 100 while, the total energy content for the NE system is 115.2, which demonstrates that the Atwater system underestimates the energy (% of calories) compared to the more accurate NE system. While the NE system is more accurate than the Atwater system for calculating true dietary energy content, it also has limitations. Ingredients vary in chemical composition among sources which makes it difficult to accurately predict true available energy, even if the NE system gives a more accurate value (Kil et al., 2013). However, the NE system is influenced by the internal and external environment of the body, which affects the NE requirement for maintenance, and therefore, any changes to the environment could potentially negatively impact the use of the NE system (Kil et al., 2013). In growing pigs, every 1˚C decrease in environmental temperature below the lower critical temperature, leads to a 4% increase in maintenance energy requirement due to an increase in body heat production to maintain appropriate body temperature (Close, 1997). The NE system has not been as well-defined as the Atwater system until recent years which may limit the confidence of nutritionists in using it (Whitney, 2017). Furthermore, the NE system can be very expensive and tedious to determine energy values of many ingredients (Whitney, 2017). In conclusion, the current systems used to calculate available energy from food sources have many limitations for accurate estimation of energy content of feed ingredients and diets. The NE system is used in livestock nutrition to provide a more accurate assessment of available energy value of feed ingredients because it accounts for heat losses from digestive and fermentation processes, especially when feeding high fiber diets. While there are challenges with using the NE system, the benefits of greater accuracy exceed these limitations compared with using the Atwater system. Therefore, all human and animal

nutrition studies would greatly benefit by using the NE system instead of the Atwater system when formulating diets.

Effect of Maternal Mineral Deficiencies on Reproduction and Growth of Rodent Offspring The National Research Council provides estimates of the minimum requirements for the amount of macro- and micro-nutrients in diets for all animal species to support optimal growth, reproduction, and health. The diets used in my thesis research study were formulated using the NE system to meet or exceed all of the mouse NRC requirements. We did not anticipate that the experimental diets we fed during this study were deficient in all minerals, which severely compromised our ability to test our original hypothesis. Therefore, a brief review of the scientific literature on the effects of mineral deficiencies in mice is relevant to this thesis. Minerals in animal nutrition are classified as macro-minerals and micro (trace) elements (Soetan et al., 2010). Elements such as calcium, phosphorus, sodium and chloride are considered macro-nutrients while iron, copper, cobalt, potassium, iodine, magnesium, zinc, manganese, fluoride, chromium, selenium and sulfur are considered micro-elements (Eruvbetine, 2003). Deficiencies in these minerals can cause a variety of metabolic diseases and challenges for the animals which are summarized in Table 3 (Soetan et al., 2010).

Table 3. Summary of macro- and micro-mineral elements required in mouse diets, major functions of these elements, and deficiency signs (adapted from Hays and Swenson, 1985; Malhotra, 1998; Murray et al., 2000; Soetan et al., 2010; Streeten and Williams, 1952). Element Major Functions Deficiency Symptoms Macro-minerals Calcium (Ca) Regulates nerve and muscle Rickets, soft deformed function, constituent of bone and bones, decalcification of

teeth bones Phosphorus (P) Constituent of bone, teeth, ATP, Rickets, nucleic acids, and buffers fluids hyperparathyroidism, De Toni-Fanconi Syndrome Sodium (Na) Principal cation in extracellular Hypoatraemia, vomiting, fluids, maintains osmotic diarrhea, intestinal pressure obstruction Chlorine (Cl) Important for optimal cation and Reduced excretion in anion balance of body fluids and urine and perspiration, secretions secondary to vomiting, renal disease, and diuretic therapy Potassium (K) Regulates osmotic pressure, Impacts collecting muscle (cardiac) contraction, tubules of kidney, alters conducts nerve impulse gastric secretions and intestinal motility, impaired neuromuscular functions of muscles Magnesium (Mg) Constituent of bones, teeth and Vasodilation with enzyme cofactor erythemia and hyperaemia Sulfur (S) Present in cysteine, cysteine and Reduced digestibility, methionine, connective tissue, feed intake, weight gain skin, nails and hair and milk production Micro-minerals Chromium (Cr) Nucleoproteins, RNA preps, Essential for rodent maintains configuration of RNA growth, reduced lifespan, molecule, cross-linking agent for reduced rate of removal collagen of ingested glucose

Cobalt (Co) Constituent of vitamin B12, Symptoms manifested in

cofactor of enzymes involved in vitamin B12 deficiency, DNA biosynthesis anorexia, fatty livers Copper (Cu) Constituent of enzymes, plays a Anemia (hypochromic role in iron absorption, and microcytic) necessary for neurologic systems Iodine (I) Basic component of thyroid Cretinism, goiter and hormones hypothyroidism and myxedema Iron (Fe) Transports oxygen, essential Anemia, impacts GABA component of enzymes involved metabolism in rats, in biological oxidation, required alterations in many for proper myelination of spinal metabolic processes that cord, synthesis and packaging of impact brain functioning neurotransmitters Manganese (Mn) Cofactor of hydrolase, Deformities of bones, decarboxylase, and transferase poor growth, and enzymes impaired reproduction Selenium (Se) Constituent of glutathione Results in white muscle peroxidase, and protects disease, disrupts normal organism from free radicals reproductive process, higher incidence of embryonic mortality Silicon (Si) One of the most abundant Reduced weight gain and elements in animal tissue, plays pathological changes in a role in connective tissue and formation and structures bones of bones and connective tissue Zinc (Zn) Cofactor of many enzymes Skin lesions and diarrhea 25

involved in cell metabolism and cell replication, nucleic acid and amino acid metabolism Fluorine (F) Provides strength to teeth Anemia, reduced fertility, enamel and increases hardness and growth retardation of bones

In mice, mineral deficiencies, specifically copper and zinc, have acute and chronic developmental consequences (Keen et al., 2003). A zinc deficiency affects nucleic acid metabolism and causes higher rates of cellular oxidative damage and apoptosis, as well as a reduction in protein metabolism and tubulin polymerization to impact embryonic and fetal development (Jankowski-Hennig et al., 2000; Keen, 1996; Mackenzie et al., 2002). In rodents, a zinc deficiency before mating and throughout pregnancy will result in poor fertilization, and offspring exhibit defects in almost every organ system (Keen, 1996; Carl L. Keen et al., 2003). Defects in response to mineral deficiencies such as zinc and copper are observed rapidly, after 48 hours of exposure to deficient diets (Hawk et al., 1998). Copper deficiency in rodents can lead to reduced fertilization rates, embryonic death, cardiovascular, central nervous system, and skeletal defects in the offspring through excessive oxidative damage, impaired oxidant-defense system and energy production, and decrease in lysyl oxidase activity (Keen et al., 1998; Menino et al., 1986; Prohaska, 2000; Rucker et al., 1998). In addition to negatively influencing reproduction of mice, mineral deficiencies (both macro- and micro-) can disrupt the programming of fetal genes which can impair the health and lifespan of the offspring throughout adulthood, and also result in poor antibody formation (Chandra, 1975; Carl L. Keen et al., 2003). Feeding a maternal diet marginally deficient in iodine results in no effect on the uptake of iodine by the maternal thyroid, but a 50% decrease in iodine uptake by the fetal thyroid (Versloot et al., 1997). This is important because a maternal diet deficient in iodine may negatively impact fetal development, especially brain development,

due to diminished thyroid hormone production. The results from all of these studies indicate that micro-minerals are critically important for the healthy fetal and embryonic development of rodents. Mineral deficiency in the absence of caloric deficiency can occur in some human and animal populations. For instance, in humans, nearly 50% of pregnant women have inadequate magnesium and iron intake, and many are deficient in vitamin D, causing life-long changes in offspring physiology (Abu-Ouf and Jan, 2015; Gupta et al., 2014; Wen et al., 2018). Magnesium restriction causes altered lipid metabolism, as well as decreased survival and growth restriction of offspring (Gupta et al., 2014). Vitamin D deficiencies are associated with increased body fat and adiposity in mice and human offspring (Wen et al., 2018). Maternal iron deficiency is correlated to elevated concentrations of fibroblast growth factor-23 in infants (Clinkenbeard et al., 2014), which is a hormone that regulates phosphate and can impact the metabolism of phosphate that may lead to compromised bone development in mice and human offspring (Braithwaite et al., 2016). Multiple reports have demonstrated that feeding selenium deficient diets to mice leads to a decrease in liver glutathione peroxidase activity (Reiter et al., 1989; Toyoda et al., 1989; Weitzel et al., 1990; Wendel and Otter, 1987). Congenital irreversible ataxia and early postnatal death in mice can be related to prenatal manganese deficiency (Erway et al., 1970; Hurley and Theriault Bell, 1974). There appear to be no published studies that have evaluated the effects of trace minerals such as chromium, cobalt, silicon, fluorine, and sulfur deficient maternal rodent diets on pregnancy and offspring growth. Calcium deficiency in rats can lead to severe hypocalcaemia in dams during lactation, reduction in long bone growth and ash content, impairment of rearing and stunted growth of offspring (Rasmussen, 1977). This means that while rats can grow and reproduce on a calcium deficient diet, they have difficulty rearing their offspring during lactation that in turn leads to smaller and more immature pups at weaning. Phosphorus deficiency can cause rickets, similar to calcium, because calcium and phosphorus are often regulated together in the

body. In rats fed vitamin D-deficient diets containing low, medium, or adequate dietary concentrations of phosphorus, rickets and osteomalacia were observed in the diet containing low levels of phosphorus while only rickets were observed in the diet containing medium levels of phosphorus, and no abnormalities were noticed in the diet containing normal levels of phosphorus (Holtrop et al., 1986). Therefore, the mineralization of bones depends on obtaining adequate amounts of calcium and phosphorus from the diet. Sodium deficiency in rats can lead to a decrease in maternal and fetal sodium plasma concentrations, which ultimately determines the osmolarity of extracellular fluids and plasma (Dancis and Springer, 1970). Maternal diets deficient in choline results in a decrease in the number of blood vessels in the fetal hippocampus, increases mRNA expression of angiogenic signaling molecules, increases protein expression of cyclin- dependent kinase inhibitors, and ultimately cause long-term changes in memory function (Mehedint et al., 2010; Niculescu, 2006). In order to raise healthy offspring, diets containing sufficient amounts of macro-minerals are required in rodents. Maternal restriction of a vitamin and mineral mixture (50% of control diet) increased epididymal fat pad weight, variable expression of adipocytokines, no insulin resistance, and with the restoration of vitamins/minerals after birth in rat offspring, these negative effects were reversed (Lagishetty et al., 2007). There appear to be no published studies examining the impact of combined multiple mineral deficiencies in maternal mouse diets in the absence of caloric restriction on offspring health. Long-term studies to determine the effects of in utero micronutrient deficiency or supplementation are needed (Gernand et al., 2016). Conclusions Over the past 50 years, there have been many advances in support of the concept of developmental programming in humans and animals. Numerous studies have been conducted to determine the various effects of maternal nutrition, especially a high fat diet on offspring growth, body composition, and metabolic health. Overall, maternal nutrition including mineral deficiencies, is

very important for the normal development and health of offspring, particularly in the case of a maternal high fat diet predisposing the offspring to metabolic disorders. There are several knowledge gaps in the scientific literature, especially involving the effects of feeding isocaloric diets and restricting the calories of a maternal high fat diet on offspring. Likewise, there is controversy of the most appropriate energy measurement systems to use when evaluating the effects of dietary calorie intake in rodent models. The use of the NE system to formulate maternal high fat diets to determine the effects of restricting these calories on developmental programming of offspring is more accurate than using the Atwater energy system.

Chapter 2: Physiological Effects of Maternal Isocaloric and Mineral Deficient Diets On Offspring Growth and Body Composition

Synopsis Over the past 30 years, many researchers have evaluated the various effects of a maternal high fat diet on the growth, body composition, and health of the offspring. However, no study has been published to evaluate if developmental programming is due to diet composition independent of body composition. We hypothesized that diet composition of maternal diets would induce growth, body composition, and physiological changes in offspring. To test this hypothesis, we fed 3 groups of mouse dams isocaloric diets, where one group was fed a low fat-restricted (LFr) diet, and two groups were fed either a high fat diet with ad libitum access (HFa), or the high fat diet that was calorically restricted (HFr) based on the feed intake of pair-fed mice on the low-fat diet. Three-weeks old parent Agouti Avy strain non-carrier wild-type mice were randomly weaned and fed either a low-fat, high-carbohydrate diet (LF, 7.5% kcal lipid from canola oil, 72.7% kcal from carbohydrates) or a high-fat, low- carbohydrate diet (HF, 31.3% kcal lipid from canola oil, 46.9% kcal carbohydrates). Parent mice were mated at eight weeks of age. The offspring were assigned to four subsequent dietary treatment groups; 1) dams fed LFr and offspring fed low fat diet with ad libitum (LFa) access (LFr:LFa), 2) dams fed LFr and offspring fed HFa (LFr:HFa), 3) dams fed HFr and offspring fed HFa (HFr:HFa), and 4) dams fed HFa and offspring fed HFa (HFa:HFa). Average weekly body weight and composition of offspring were measured in individual mice in each of the four dietary treatment groups. Unfortunately, the experimental diets obtained from the manufacturer were deficient in minerals resulting in high pup mortality during lactation and post-weaning, which prevented us from testing our original hypothesis. Overall, dams (24 weeks of age) and offspring (12 weeks

of age) fed the low fat diet had the greatest body weight gain and average percentage of body fat mass than those fed the other dietary treatments. The physiological changes in growth and body composition observed were opposite of the results we expected, or were not significantly different among dietary treatments. In conclusion, feeding the mineral deficient diets confounded the effects of dietary energy responses. This study should be repeated to determine the effects of dietary calorie source and consumption on developmental programming in mice.

Introduction Over 70% of men and women in the United States are overweight or obese, according to the NHANES data from 2013-2014 (Centers for Disease Control and Prevention, 2016). Obesity can be caused by diet, lack of exercise, maternal age, genetics, and epigenetics, which is a change in gene expression without altering the underlying DNA sequence (McAllister et al., 2009). Among the many factors that impact offspring weight, maternal caloric restriction and feeding a maternal high fat diet may play an important role. Recently, several studies have evaluated the various effects of feeding a maternal high fat diet on offspring longevity and metabolic health. In a review by Williams et al. (2014), the normal functions of several organs and systems in offspring, including heart, bone, gut microbiome, pancreas, skeletal muscle, placenta, adipose tissue, liver, vasculature, and brain, can be decreased by feeding a maternal high fat diet. Females exposed to caloric and nutrient restriction during early gestation have been shown to have increased rates of adult obesity, while females fed deficient diets during mid-pregnancy had biomarkers indicating reduced renal function (Schulz, 2010). Mouse offspring fed a control diet that were produced from dams consuming a high fat diet (16% fat, 33% simple sugars, energy 3.5 kcal/g), had increased body weight, metabolic and cardiovascular disease, hypertension, adipocyte metabolism and insulin resistance when compared with offspring from dams fed a control diet (3% fat,

7% simple sugars, energy 4.5 kcal/g; Samuelsson et al., 2008). Dams fed a high fat diet (45% calories from lipids/100g of diet, 4.6 kcal/g) had 11% greater body weight during gestation than dams fed the control diet (9% calories from lipids/100g of diet, 3.8 kcal/g (Ashino et al., 2012). Mouse offspring from dams that were fed a high-fat diet during pregnancy exhibited metabolic syndrome-like characteristics which included obesity, high blood pressure, high concentrations of blood glucose and serum triglycerides, low concentrations of high-density lipoprotein, which are associated with the risk for developing cardiovascular disease and type-2 diabetes (Masuyama and Hiramatsu, 2012). Although previous research studies have evaluated the effects of feeding maternal high fat diets on offspring, it is still unknown if the documented effects are due to caloric intake or calorie source. It is also unknown if feeding maternal high fat diets causes weight differences based on the proportional increase in body size or due to differences in body composition. All of the diets in previously reported mouse studies were formulated using metabolizable energy based on Atwater values, which is a common method used when formulating rodent diets (Bielohuby et al., 2010). The Atwater system does not account for the digestibility and subsequent utilization of energy and nutrients of the ingredients and diets of mice, therefore this is an inaccurate approach for formulating diets. Formulating diets for food producing animals involves using more advanced and accurate measures of energy and digestible nutrients to meet nutritional requirements. Therefore, interpreting and comparing nutritional responses among mouse studies is difficult because of dramatic differences in energy values of dietary ingredients used, and their inclusion rates. Therefore, using the NE system, which is the system used to formulate diets for food producing animals, would be more accurate for mouse studies, especially in those evaluating the effects of feeding isocaloric diets. Even if there are modest differences between the two systems, the NE system provides more accurately formulated diets.

Cannon et al. (2014) observed that six-month old male mice from dams that consumed a low-fat-high-carbohydrate diet that were subsequently fed a high-fat-low-carbohydrate diet, weighed more than offspring mice fed a high-fat- low-carbohydrate diet when their dams consumed the same high-fat-low- carbohydrate diet. Both diets were formulated to be isocaloric, by adding pectin and cellulose to the high fat diet, to avoid confounding due to differing caloric intakes. However, confounding of dietary treatment responses still occurred because the dams were fed a high fat diet ad libitum resulting in obesity or excessive body weight. In fact, all of the maternal high fat diet studies provided parents with ad-libitum access to diets during gestation and lactation, which led to excessive body weight and obesity. Due to the confounding factor of obesity, the claim that maternal high fat diets influence development of offspring needs to be evaluated to determine if the responses occur due to maternal body weight and obesity or from maternal calorie source. If the calorie consumption from feeding a high fat diet were controlled or restricted to provide maternal body composition similar to that of dams fed a low fat diet, the confounding effects of maternal body weight and obesity could be independently determined from maternal caloric intake responses. Therefore, the objective of this study is to determine if diet composition alone, excluding the confounding factor of body composition, induces intergenerational changes in offspring growth, body composition, and physiological changes. We hypothesize that diet composition will induce intergenerational changes in offspring growth and body composition.

Materials and Methods To address the objective of determining if intergenerational changes in offspring growth and body composition occur in response to dietary calorie source and intake, 3 groups of mouse dams and sires were fed isocaloric diets based on the net energy (NE) system for diet formulation. One group was fed a controlled amount of a low-fat diet daily (LFr) in order to control the total amount of calories consumed (average energy consumed weekly over 11 weeks was

18.26 calories using NE formulation), and two groups were fed either a high fat diets that was provided ad libitum (HFa) or a high-fat diet that was pair-fed daily to restrict calorie intake comparable to the feed intake of the low-fat diet (HFr, average calories consumed weekly over 11 weeks was 18.26 calories using NE formulation) as shown in figure 2.

F0 mice (dams and sires for F1)

Weaning

Low fat diet High fat diet High fat diet restricted amount restricted amount fed ad libitum fed daily fed daily

Group 1 Group 2 Group 3 (LFr) (HFr) (HFa)

Figure 2. Experimental design of dietary treatments of parent (F0) mice.

Animals Agouti Avy strain non-carrier wild-type mice (genetically invariant background that is 93% similar to C57Bl/6j) from an existing breeding colony were mated to produce the F0 generation (Faulk et al., 2014; Waterland and Jirtle, 2003; Weinhouse et al., 2014). Mice were housed in a 22° C temperature- controlled room using a sustained 12-hour light-dark cycle, and were provided ad libitum access to water. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (protocol # 1704-34760A).

Experimental Design

A total of 22 females and 24 male three-week old mice were used as the parents of the F0 generation, and were tail-tipped, individually housed, randomly weaned, and assigned to either a low-fat, high-carbohydrate diet (LF, 7.5% kcal from lipid, 72.7% kcal from carbohydrates) or a high-fat, low-carbohydrate diet (HF, 31.3% kcal from lipid, 46.9% kcal from carbohydrates; Table 4, 5 and 6).

Table 4. Calculated nutrient composition of the low fat and high fat experimental diets. Nutrient Low Fat Diet High Fat Diet Crude protein, % 18.3 18.2 Indispensable amino acids, % Histidine 0.52 0.52 Isoleucine 0.96 0.96 Leucine 1.73 1.73 Lysine 1.45 1.45 Methionine 0.52 0.52 Phenylalanine 0.96 0.96 Threonine 0.77 0.77 Tryptophan 0.22 0.22 Valine 1.14 1.14 Dispensable amino acids, % Alanine 0.55 0.55 Arginine 0.70 0.70 Aspartic Acid 1.29 1.29 Cystine 0.37 0.37 Glutamic Acid 4.08 4.09 Glycine 0.39 0.39 Proline 2.36 2.36 Serine 1.10 1.10

Taurine 0.00 0.00 Crude fat, % 3.1 11.6 Cholesterol, mg/kg 0.00 0.00 Linoleic acid, % 0.63 2.42 Linolenic acid, % 0.30 1.15 Arachidonic acid, % 0.00 0.00 Omega-3 fatty acids, % 0.30 1.15 Total saturated fatty acids, % 0.24 0.92 Total monounsaturated fatty 1.80 6.91 acids, % Polyunsaturated fatty acids, % 0.87 3.35 Fiber (max) , % 5.0 25.0 Carbohydrates1, % 67.2 39.1 Macro minerals, % Calcium2 0.51 0.50 Phosphorus2 0.32 0.32 Potassium2 0.36 0.36 Magnesium2 0.05 0.05 Sodium2 0.14 0.13 Chloride2 0.23 0.21 Micro minerals, mg/kg Fluorine2 1.00 1.00 Iron2 40.00 40.00 Zinc2 35.00 35.00 Manganese2 11.00 11.00 Copper2 6.00 6.00 Iodine2 0.21 0.21 Chromium2 (added) 1.00 1.00 Molybdenum 0.14 0.14

Selenium2 0.24 0.24 Vitamins Vitamin A3, IU/g 4.00 4.00 3 Vitamin D3 (added), IU/g 1.00 1.00 Vitamin E3, IU/kg 75.00 75.00 Vitamin K3, mg/kg 0.75 0.75 Thiamin hydrochloride, mg/kg 4.80 4.80 Riboflavin, mg/kg 6.70 6.70 Niacin, mg/kg 30.00 30.00 Pantothenic acid, mg/kg 16.00 16.00 Folic acid, mg/kg 2.10 2.10 Pyridoxine, mg/kg 5.80 5.80 Biotin, mg/kg 0.20 0.20 3 Vitamin B12 , µg/kg 28.00 28.00 Choline chloride, mg/kg 1,250 1,250 1Corn starch was used as the source of carbohydrates 2Sources of minerals were as follows: Ca, calcium carbonate, anhydrous 40.04%; P, potassium phosphate monobasic 22.76%; K, potassium citrate tri-potassium monohydrate 36.16%; Mg, magnesium oxide 60.32%; Na, sodium chloride 39.34%; Cl, sodium chloride 60.66%; F, sodium fluoride 45.24%; Fe, ferric citrate 16.50%; Zn, zinc carbonate 52.14%; Mn, manganous carbonate 47.79%; Cu, cupric carbonate 57.47%; I, potassium iodate 59.30%; Cr, chromium potassium sulfate 10.42%; Se, sodium selenate, anhydrous 41.79%. 3Sources of vitamins were as follows: vitamin A, all-trans-retinyl palmitate

500,000 IU/g; vitamin D3, cholecalciferol 400,000 IU/g; vitamin E, all-rac-a- tocopheryl acetate 500 IU/g; vitamin K, phylloquinone; vitamin B12, cyanocobalamin 0.1% in mannitol. Table 5: Ingredient composition (%) of the low fat and high fat experimental diets.

Ingredient, % Low Fat Diet High Fat Diet Corn Starch 43.75 28.23 Casein 20.00 20.01 Pectin - 15.00 Canola Oil 3.00 11.51 Powdered Cellulose 5.00 10.00 Maltodextrin 13.20 6.20 Sucrose 10.00 4.00 AIN 93G Mineral Mix1 3.50 3.50 AIN 93 Vitamin Mix2 1.00 1.00 L-Cystine 0.30 0.30 Choline Bitartrate 0.25 0.25 t-Butylhydroquinone 0.0014 0.0014 1 Calcium carbonate, sucrose, potassium phosphate, sodium chloride, potassium citrate, tribasic monohydrate, potassium sulfate, magnesium oxide, ferric citrate, zinc carbonate, sodium metasilicate, manganese carbonate, cupric carbonate, chromium potassium sulfate, boric acid, sodium fluoride, nickel carbonate, lithium chloride, potassium iodate, sodium selenate, ammonium molybdate, ammonium vanadate. 2 Sucrose, DL-alpha tocopheryl acetate (form of vitamin E), phyliquinone (form of vitamin K), nicotinic acid, calcium pantothenate, vitamin A palmitate, pyridoxine hydrochloride, thiamin, hydrochloride, riboflavin, vitamin B-12 supplement, folic acid, cholecalciferol, biotin. Table 6: Comparison of the calculated energy content using the Atwater and net energy (NE) systems1, and chemical components in the low fat and high fat experimental diets. Energy Low fat diet High fat diet source Atwater, Atwater NE, NE2, Atwater, Atwater NE, NE2, kcal/g Energy2, kcal/g % kcal/g Energy2, kcal/g % % % Crude protein 0.733 19.8 0.720 23.0 0.727 21.8 0.723 23.1

Ether extract 0.278 7.5 0.188 6.0 1.041 31.3 0.729 23.3 Carbohydrates 2.688 72.7 - - 1.563 46.9 - - Digestible - - 2.676 85.5 - - 1.539 49.2 carbohydrates Fermentable - - 0.116 3.7 - - 0.570 18.2 carbohydrates Total energy, 3.70 100 3.13 118.2 3.33 100 3.13 113.8 kcal/g 1 Atwater calculations using ((4 Kcal/g * protein) + (9 Kcal/g * fat) + (4 Kcal/g * carbohydrate)). NE calculations using (NE(MJ/kg) = (11.3*digestible crude protein + 35*digestible crude fat + 14.4*digestible crude fiber + 12.1*digestible nitrogen-free extractives – 0.63*sugars)/1000). 2 % of total calories The experimental diets were formulated by TestDiet (Land O’ Lakes, Inc., St. Louis, MO) to meet the mouse NRC (1995) requirements, and were isocaloric when using the NE system for diet formulation. The amount of total energy for the low fat and high fat diets when using the NE system was the same between the diets and less than the total energy calculated using the Atwater system. One assumption of the NE system is that the proportion of net energy content relative to the total gross energy in the food will be used for maintenance and productive purposes. However, total caloric intake is also dependent on food intake. Male and female parent mice (F0) were individually housed and randomly assigned into one of three dietary treatment groups at weaning: low-fat restricted (LFr), high-fat restricted (HFr), or high fat ad libitum (HFa). There were 8 males in each group, and 8 females in the LFr, 6 females in HFr, and 8 females in HFa. All mice in the HFr group was pair-fed to the LFr group, with both of these groups being fed daily (up to 47.29 calories per day during lactation) while mice in the HFa group were provided fresh food weekly to ensure ad libitum access. Daily rations in the restricted/controlled feeding treatments (LFr and HFr) were based on a previous study that measured caloric intake within this strain using a previous grain-based control mouse diet (Teklad Global Diet 2018, Envigo, North 39

America). We used the 2018 diet to obtain estimated daily feed intake values for our LFr and HFr daily rations because the 2018 diet is a very standard mouse chow, and daily consumption of this diet were similar to those reported in NRC (1995). The daily food intake values (g) of mice fed the 2018 diet were multiplied by the total energy (kcal/g) determined by the NE system for the low fat diet. The total NE calories consumed by mice fed the low fat diet were used to estimate the daily feed intake required to match total calorie consumption for the HFr group. The amount of food provided to the LFr and HFr mice compared to the amount of food consumed is shown in Figure 3, and the daily calories offered and consumed by the three maternal dietary treatments is shown in Figure 4. The mice in the low fat and high fat restricted groups consistently did not eat all of the food that was provided to them daily. This amount was not weighed due to the fact that it is typical for mice to not eat all of the food a few days after weaning due to the stress of weaning and some might not have fully developed teeth at that age. However, the mice continued to leave food throughout the study, which indicates that something was amiss with the measured amounts that were given to the mice.

20 Low Fat Restricted High Fat Restricted 15 High Fat Ad lib 10

0 0 5 10 15 Weeks on Diet Amount of Food Given or Consumed (g)

Figure 3. Daily amount of food provided to female mice fed low fat restricted (LFr) and high fat restricted (HFr) diets compared with the amount of food (g) consumed by the female mice fed the high fat ad libitum (HFa) dietary treatment.

60 Low Fat Restricted High Fat Restricted 40 High Fat Ad lib

0 0 5 10 15

Daily Calories Offered or Consumed Weeks on Diet

Figure 4. Daily amount of calories offered to female mice fed low fat restricted (LFr) and high fat restricted (HFr) diets compared with the amount of calories consumed by the female mice fed the high fat ad libitum (HFa) dietary treatment.

The amount of calories from NE that were consumed daily were similar between the LFr and HFr treatment groups. Furthermore, the amount of calories that mice on the HFa dietary treatment consumed was similar to values reported in the 0.75 mouse NRC (12.7 kcal ME/BWkg /day for days 21 to 42) (1995). We used the NE system because the Atwater system over- and underestimates the accurate amount of energy required, therefore the mouse NRC, is out of date and inaccurate (figure 5).

50 HFa Net Energy Calories 40 HFa Atwater Calories 2018 Atwater Calories 30

10 Calories Consumed

0 1 2 3 4 5 6 7 8 9 10 11 Weeks on Diet

Figure 5. Comparison of the weekly calorie consumption by female mice fed the 2018 diet (Atwater calories), the high fat ad libitum diet (HFa; Atwater calories) and HFa (NE calories system calculations).

At eight weeks of age, each male was placed in the same cage as one female of the same diet group for mating. Males remained in the mating cage until vaginal plug was present in the females, or for a maximum of one week. After males were removed from the mating cage, they were provided ad libitum access to their assigned experimental diets as shown in Figure 6.

st High pup mortality, 1 parity, all offspring euthanized nd 2 parity

F0 F0 F0 F1.1 F1.1 F1.2 F1.2 birth weaning mating birth weaning birth weaning

3 weeks 5 weeks 3 weeks 3 weeks 3 weeks 3 weeks Fed assigned experimental diets weigh weigh weigh maturity … weaning

9 weeks

F 1.2 Males switched to ad libitum test diets, weights discontinued weigh weigh weigh weigh weigh weigh weigh weigh weigh weigh

Figure 6. Timeline of significant events that occurred throughout the study.

A total of 135 F1 offspring mice were born alive, and nursed their dams until weaning on day 21. At weaning, all offspring were provided ad libitum access to their respective experimental diets. Offspring from the HFr and HFa dams were fed the HF diet. Offspring from LFr dams were split into two groups where one group was fed the LFa diet and the other group was fed the HFa diet. Pups were unexpectedly small in body weight (< 7 g) compared to normal mice at weaning (>7 g), and were likely too immature to survive on their own by day 21, resulting in a high pup mortality rate during lactation (18% from LFr dams, 6.08% from HFr dams, and 2.38% from HFa dams) and after weaning (50% mortality rate between all treatments). As a result, all surviving pups were euthanized and the dams were re-mated to repopulate offspring numbers to produce the F1.2 generation (second parity).

During the second lactation there was a high pup mortality rate among all dietary treatment groups throughout the 21 days of lactation period (20% from LFr dams, 29.6% from HFr dams, and 23.8% from HFa dams). A total of 53 males and 42 females in the F1.2 generation were weaned once they achieved a minimum body weight of 7 g, were tail-tipped, and fed either the low fat or high fat dietary treatments consisting of 1) LFr:LFa, 2) LFr:HFa, 3) HFr:HaF, and 4) HFa:HFa (Figure 7; where the first diet abbreviation represents the dam diet and the second diet abbreviation represents the offspring diet). There were 23 pups (males n = 13, females n = 10) in the LFr:LFa group, 16 (males n = 9, females n = 7) in the LFr:HFa group, 28 (males n = 19, females n = 9) in the HFr:HFa group and 28 (males n = 12, females n = 16) in the HFa:HFa group. All animals were weighed weekly.

Group 1 Group 2 Group 3 (LFr) (HFr) (HFa)

F0 mice (dams and sires for F1)

F1.2 mice

Offspring Offspring Offspring Offspring fed low fed high fed high fed high fat fat diet fat diet fat diet diet (LFr:LFa) (LFr:HFa) (HFr:HFa) (HFa:HFa)

All offspring were fed ad libitum

Figure 7. Experimental design of dietary treatments of F1.2 mice offspring (first diet abbreviation represents the dam diet and the second diet abbreviation represents the offspring diet).

Diet Nutrient Analysis

Due to the unexpectedly high pup mortality observed in this study, we consulted with TestDiet (Land O’ Lakes, Inc., St. Louis, MO), the manufacturer of the diets used in this study, to verify that the macronutrients of the low fat and high fat diets were adequate. The analytical laboratory at TestDiet (Land O’ Lakes, Inc., St. Louis, MO) was only able to evaluate the percentage of protein, fat, fiber, carbohydrates, moisture, and ash in addition to the energy (kcal/g) and amount of calories from fat (kcal/g). Since the analytical laboratory could not analyze micronutrients, samples of the low fat and high fat diets were submitted to the soil testing laboratory at the University of Minnesota for mineral analysis to determine if they contained mineral concentrations similar to formulated concentrations. Samples underwent microwave digestion and ICP-OES analysis to determine amounts of minerals present using method EPA 3051 (10mL HNO3, heated to 180˚C; (Environmental protection agency, 2007).

Body Composition Analysis and Tissue Collection After all offspring were weaned, the dams underwent body composition analysis via EchoMRI 3-in-1 (Echo Medical Systems LLC, Houston, TX). After body composition analysis, vaginal smears were collected and analyzed to determine the estrus cycle of each female in order to perform timed euthanasia. After euthanasia, tail tip, ovaries, uterus, liver, and oviduct samples were collected from each dam. Once the offspring reached 12 weeks of age, they underwent body composition analysis via EchoMRI and were euthanized. Samples of liver, kidney, brain, tail, blood, cecum, colon, cecum contents, and colon contents were collected and snap frozen from all offspring for future microbiome and molecular physiology analysis. Samples of liver were also collected and placed in formaldehyde for future analysis or frozen for histological analysis. The frozen liver samples were sectioned using a cryostat microtome and slides were stained with fat, Oil Red O, propylene glycol stain kit (Newcomer Supply, Middleton, WI)

to evaluate liver lipid content among dietary treatment groups. Samples of ovaries and uterus from the female offspring were collected and snap frozen in liquid nitrogen. Sperm were collected from the F0 males via the “swim up” procedure (Jameel, 2008; Younglai et al., 2001) and confirmed for 99% purity, and frozen at -80° C. A frozen sample of liver for histological analysis was collected from all offspring and one dam on the high fat diet, which served as a high fat control. After all liver samples were sectioned, placed on slides, and stained with Oil Red O stain (Newcomer Supply, Inc.) to determine potential liver lipid content differences among dietary treatment groups. Fat cells were identified by the red stain and nuclei were identified by the counterstain (Hematoxylin stain) which was displayed as a blue color on the slide. Fatty livers were characterized by large amounts of red fat cells while normal livers were characterized by large amounts of blue nucleated cells.

Statistical Analysis The statistical model for this experiment was a completely randomized design with the fixed effect of diet and random effects of body weight and body composition. Normality for all data was tested using D’Agostino and Pearson tests. All data were analyzed using ANOVA followed by Tukey’s multiple comparisons test using Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA) software. A p-value < 0.05 was considered significant. A power analysis indicated that a 20% difference could be detected with 80% power by using a minimum of 10 animals per group in the F1 generation. It was assumed that a minimum of 40 animals for F1 generation would be produced by 12 animals in the F0 generation.

Results and Discussion Body Weight

Parental (F0) mice were weighed weekly and there were no differences in the average dam body weight (grams) among the 3 dietary treatment groups during the 22 week feeding period (Figure 8).

Dams Weight 50 Low Fat 40 High Fat Restricted High Fat Ad lib 30

20 Weight (g) Weight 10

0 0 5 10 15 20 25 Weeks on Diet

Figure 8. Average weekly body weight (g) of the dams fed low fat restricted (LFr; n = 8), high fat restricted (HFr; n = 6), of high fat ad libitum (HFa; n = 8) diets (P = 0.98).

For the F1 offspring, it was expected that those produced from dams fed the low fat diet and fed the high fat offspring diet (LFr:HFa) would have the greatest average body weight, followed by offspring from dams fed the high fat diet ad libitum when offspring were fed the high fat ad libitum diet (HFa:HFa). Offspring from dams fed the high fat restricted diet and fed the high fat ad libitum diet (HFr:HFa), and those from dams fed the low fat restricted diet and subsequently fed the low fat offspring diet (LFr:LFa) were expected to be the groups that would have had the lowest average body weight. However, male offspring the LFr:LFa group had the greatest final body weight after 10 weeks, followed by those fed LFr:HFa, with the HFr:HFa and HFa:HFa groups having the least body weight (Figure 9).

Male Offspring 40 LFr:LF LFr:HF 30 HFr:HF 20 HFa:HF Weight (g) Weight 10

0 0 5 10 15 Weeks on Diet

Figure 9. Average weekly body weight (g) of the male offspring among the four maternal and offspring dietary treatment combinations with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 9), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 13), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 19), and HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 12; P = 0.27).

These results were not expected because there were no differences in body weight among the four groups of male offspring. Similarly, there were no differences (P > 0.11) among the four groups for the female offspring with the HFr:HFa group weighing the least of all the groups (Figure 10).

Female Offspring 25 LFr:LF 20 LFr:HF HFr:HF 15 HFa:HF 10 Weight (g) Weight 5

0 0 5 10 15 Weeks on Diet

Overall, there were no differences in male and female offspring body weights during a 10 week feeding period for fed any of the dam and offspring treatment combinations, which was contrary to our expectations. We expected to observe that dams fed HFa would have the greatest body weight, followed by those fed HFr and LFr, with dams in these two groups having similar body weight at 24 weeks of age. For the offspring, we expected that those from the LFr:HFa treatment to have the greatest body weight, followed by those in the HFa:HFa treatment at 12 weeks of age. Finally, we expected that the LFr:LFa and HFr:HFa groups would have the least body weight based on the results reported by Cannon et al. (2014). In contrast to the expected results, we observed similar average body weights between the LFr, HFr, and HFa dams.

There are several potential reasons for the results obtained in this study. First, we fed diets formulated to be similar to a human high fat diet rather than a more extreme typical high fat rodent diet. An extreme high fat rodent diet typically contains about 60% of calories from fat while our high fat diet only contained ~30% of calories from fat (Krasnow et al., 2011; Peleg-Raibstein et al., 2012; Vucetic et al., 2015). The unexpected weight gain patterns among the dietary treatment groups in our study may have been due to the moderately high fat content compared with the much greater dietary fat content used in the Cannon et al. (2014) study. However, Elahi et al. (2009) fed mice a diet containing only 18% of calories from fat (animal lard) and observed an increase in body weight compared with mice fed a control diet. Secondly, offspring body weights tended to diverge near sexual maturity in previous studies, and the shorter amount of time in which our study was conducted may not have provided enough time for differences to occur (Riska et al., 1984). However, other studies also showed no differences in body weight between mice fed high fat diets and control diets, which suggests that our results may not be unusual (Chechi and Cheema, 2006; Franco et al., 2012). Finally, our diets were deficient in macro and micro minerals, which we suspect was the primary cause for these unexpected results.

Body Composition The absolute grams of fat, fat-free mass, and free water were summed for each mouse to estimate body weight. The percentage of fat and fat-free mass was calculated by dividing the absolute grams of fat or fat-free mass per mouse by the estimated body weight per mouse, and multiplied by 100. At 24 weeks of age, the average percentage of fat of the (F0) dams ranged from 9.96% ± 1.05 to 17.77% ± 2.33. The average percentage of fat-free mass of the dams ranged from 81.79% ± 2.27 to 89.50% ± 1.05. The dams fed LFr had a greater average percentage of body fat compared with dams fed HFr (P = 0.003) and HFa (P = < 0.0001). Dams fed LFr had less average percentage of fat-free body mass compared with dams fed HFr (P = 0.005) and HFa (P < 0.0001; Figure 11A & B). 50

A. B. Dams - Fat Dams - Fat Free Mass

25 100 a Low Fat b a a Low Fat 20 b High Fat Restricted 80 High Fat Restricted High Fat Ad Lib High Fat Ad Lib 15 b 60

% Fat 10 40

5 % Fat Free Mass 20

0 0

Low Fat Low Fat

High Fat Ad Lib High Fat Ad Lib High Fat Restricted High Fat Restricted Figure 11. Average percentage of body fat (A) and fat-free mass (B) of dams at 24 weeks of age among the three maternal dietary treatments with LFr representing low fat restricted diet (n = 8), HFr representing high fat restricted diet (n = 6), and HFa representing high fat ad libitum diet (n = 8). Different letters indicate statistically significant differences (P < 0.05).

At twelve weeks of age, the average percentage of body fat in the offspring (F1.2 generation) ranged from 4.53% ± 1.71 to 12.92% ± 2.79. The average percentage of fat-free body mass of the offspring ranged from 86.78% ± 2.77 to 94.87% ± 1.70. The offspring in the LFr:LFa group had greater average percentage of body fat compared with those from LFr:HFa (P = < 0.0001), HFr:HFa (P = < 0.0001), and HFa:HFa (P = < 0.0001) groups. In addition, offspring in the LFr:HFa group had greater average percentage body fat than those in the HFa:HFa group (P = 0.0001), and offspring in the HFr:HFa group had greater average percentage of body fat than those in the HFa:HFa group (P = 0.04). Offspring in the LFr:HFa group had less percentage of fat-free body mass than those in the HFa:HFa group (P = 0.0005), and LFr:LFa offspring had less percentage of fat-free mass than those in the LF:HFa (P = < 0.0001), HFr:HFa (P = < 0.0001), and HFa:HFa (P = <0.0001) groups. Furthermore, offspring in the HFr:HFa group had less percentage of fat-free body mass than those in the HFa:HFa group (P = 0.03; Figure 12A & B). 51

B. A.

Pups - Fat Pups - Fat Free Mass

20 150 Low Fat: Low Fat Low Fat: Low Fat a Low Fat: High Fat 15 c b, c b a Low Fat: High Fat High Fat R: High Fat 100 High Fat R: High Fat 10 b b High Fat Ad High Fat Ad % Fat c 50 5 % Fat Free Mass 0 0

High Fat Ad High Fat Ad

Low Fat: LowLow Fat Fat: High Fat Low Fat: LowLow Fat Fat: High Fat High Fat R: High Fat High Fat R: High Fat Figure 12. Average percentage of body fat (A) and fat free mass (B) of offspring at 12 weeks of age among the four maternal and offspring dietary treatment combinations with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 16), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 23), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 28), and HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 28). Different letters indicate statistically significant differences (P < 0.05).

At twelve weeks of age, the average percentage of body fat of female offspring (F1.2 generation) ranged from 4.92% ± 2.01 to 13.11% ± 2.33, and the average percentage of fat-free body mass ranged from 86.61% ± 2.31 to 94.48% ± 1.96. The average percentage of body fat in the male offspring (F1.2 generation) ranged from 4.02% ± 1.09 to 12.78% ± 3.18, with the average percentage of fat-free body mass ranging from 86.91% ± 3.16 to 95.40% ± 1.15. Male offspring in the LFr:HFa group had less average percentage of body fat than those in the LFr:LFa group (P = < 0.0001) and female offspring in the LFr:LFa group (P = < 0.0001). Female offspring in the LFr:HFa group had less average percentage of body fat than male offspring in the LFr:LFa group (P = 0.0006) and the female offspring in the LFr:LFa group (P = 0.0004). Female offspring in the LFr:HFa group had a greater average percentage body fat than 52

the male offspring in the HFr:HFa (P = 0.02) and HFa:HF groups (P = < 0.0001), and the female offspring in the Hfa:HFa group (P = 0.0007). Male offspring in the LFr:LFa group had more average percentage of body fat than those in the HFr:HFa (P = <0.0001) and HFa:HFa (P < 0.0001), and female offspring in the HFr:HFa (P = <0.0001)and HFa:HFa (P = < 0.0001) groups. Female offspring in the LFr:LFa group had more average percentage of body fat than those in the HFr:HFa group (P < 0.0001), HFa:HFa group (P < 0.0001), and male offspring in the HFr:HFa group (P < 0.0001), and HFa:HFa group (P < 0.0001; Figure 13).

Offspring (Males vs Females) - Fat

20 Low Fat: Low Fat Males a a 15 Low Fat: Low Fat Females Low Fat: High Fat Males b 10 b, c c c Low Fat: High Fat Females % Fat c High Fat R: High Fat Males c 5 High Fat R: High Fat Females High Fat Ad Males 0 High Fat Ad Females

High Fat Ad Males High Fat Ad Females Low Fat: Low LowFat MalesFat: High Fat Males Low Fat: Low LowFat FemalesFat:High High Fat Fat R: FemalesHigh Fat Males High Fat R: High Fat Females Figure 13. Average percentage of fat composition between male and female offspring among the four maternal and offspring dietary treatment groups with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 9 males and 7 females), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 13 males and 10 females), HFr:HFa representing the high fat restricted dam diet, high fat ad libitum offspring diet (n = 19 males and 9 females), HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 12 males and 16 females). Different letters indicate statistically significant differences (P < 0.05). 53

Female offspring in the LFr:HFa group had less average percentage of fat free body mass than those in the HFa:HFa group (P = 0.02) and male offspring in the HFa:HFa group (P = 0.002). Male offspring in the LFr:LFa group had less average percentage of fat free body mass than those in the LFr:HFa group (P < 0.0001), HFr:HFa group (P < 0.0001), HFa:HFa group (P < 0.0001) and female offspring in the LFr:HFa group (P = 0.01), HFr:HFa group (P <0.0001), and HFa:HFa group (P < 0.0001). Female offspring in the LFr:LFa group had less average percentage of fat free body mass than those in the LFr:HFa group (P = 0.01), HFr:HFa group (P < 0.0001), HFa:HFa group (P < 0.0001) and male offspring in the LFr:HFa group (P < 0.0001), HFr:HFa group (P < 0.0001), and HFa:HFa group (P < 0.0001; Figure 14).

Offspring (Males vs. Females) - Fat Free Mass 150 Low Fat: Low Fat Males Low Fat: Low Fat Females a,c c a,c a,c a a 100 b b Low Fat: High Fat Males Low Fat: High Fat Females

50 High Fat R: High Fat Males High Fat R: High Fat Females % Fat Free Mass High Fat Ad Males 0 High Fat Ad Females

High Fat Ad Males High Fat Ad Females Low Fat: Low LowFat MalesFat: High Fat Males Low Fat: Low LowFat FemalesFat:High High Fat Fat R: FemalesHigh Fat Males High Fat R: High Fat Females Figure 14. Average percentage of fat free mass composition between male and female offspring among the four maternal and offspring dietary treatment groups with LFr:HFa representing the low fat restricted dam diet and high fat ad libitum offspring diet (n = 9 males and 7 females), LFr:LFa representing the low fat restricted dam diet and low fat ad libitum offspring diet (n = 13 males and 10 females), HFr:HFa representing the high fat restricted dam diet, high fat ad 54

libitum offspring diet (n = 19 males and 9 females), HFa:HFa representing the high fat ad libitum dam diet and high fat ad libitum offspring diet (n = 12 males and 16 females). Different letters indicate significant differences (P < 0.05).

Overall, when comparing the body composition differences of the dams among the dietary treatment groups, we expected to observe greater percentage of body fat mass in the HFa group, with the LFr group having the least. In contrast, we observed that dams fed LFr had a greater percentage of body fat than dams fed HFr and HFa at 24 weeks of age (P = 0.004 and < 0.0001, respectively). Our results may be due to feeding moderately high fat diets compared with those fed in other studies. However, Elahi et al. (2009) fed a high fat diet containing 18% of calories from fat, which was less than our diets, and observed an increase in percentage of body fat in the offspring, both males and females. Similarly, in the offspring, we expected the LFr:HFa group to have the greatest percentage of body fat mass followed by the HFa:HFa, HFr:HFa and LFr:LFa groups. However, at 12 weeks of age, the offspring in the LFr:LFa group had the greatest percentage of body fat mass, which was similar to that of the LFr dams, followed by offspring in the LFr:HFa and HFr:HFa groups. Offspring in the HFa:HFa group had the lowest percentage of fat mass. Again, these results were unexpected and may be attributed to offspring from the dams fed the high fat maternal diet requiring a more time to mature than the offspring produced from dams fed the low fat maternal diet. We are unaware of any studies in the literature that show that feeding a maternal high fat diet results in producing immature offspring. Several studies have measured maternal and offspring body composition in response to feeding a maternal high fat diet, and results from these studies have consistently shown an increase in body fat mass compared to feeding maternal control diets (Bruce et al., 2009; Cannon et al., 2014; Elahi et al., 2009; Franco et al., 2012; Hartil et al., 2009; Huypens et al., 2016a; Krasnow et al., 2011; Li et al., 2015; Volpato et al., 2012; Wu et al., 1998). These

unexpected responses were likely due to multiple dietary macro and micro mineral deficiencies, which will be subsequently described in detail.

Histology The liver from the dams fed the high fat maternal diet did not contain a large amount of red stained fat cells, indicating that the liver was considered normal, and not a fatty liver as expected (Figure 15).

Figure 15. Histology of a liver section from a dam fed the high fat maternal diet stained with Oil Red O stain for fat content at 10x magnification.

The livers from the offspring in the HFa:HFa and HFr:HFa groups did not contain a large amount of red fat cells, indicating that the offspring of both groups had normal livers, not a fatty liver as expected (Figure 16 A & B).

A. B.

Figure 16. Histology of a liver section from the offspring fed the maternal and offspring dietary treatment group HFa:HFa (A) representing the high fat ad libitum dam diet and high fat ad libitum offspring diet and HFr:HFa (B) representing high fat restricted maternal diet and high fat ad libitum offspring diet stained with Oil Red O stain for fat content at 10x magnification.

The livers from the offspring in the LFr:HFa group did not contain a large number of red stained fat cells indicating that the offspring from the LFr:HFa group had normal livers, not a fatty liver as expected (Figure 17 A). Furthermore, the livers from the offspring in the LFr:LFa group did contain a large amount of red fat cells indicating that the offspring from the LFr:LFa group did have fatty livers, not a normal liver as expected (Figure 17 B). A. B.

Figure 17. Histology of a liver section from the offspring fed the maternal and offspring dietary treatment group LFr:HFa (A) representing the low fat restricted dam diet and high fat ad libitum offspring diet and LFr:LFa (B) representing low fat restricted maternal diet and low fat ad libitum offspring diet stained with Oil Red O stain for fat content at 10x magnification. Our unexpected results may have been due to the diets not containing a greater amount of fat or feeding mineral deficient diets, which were later discovered and may have altered lipid metabolism. Many studies have evaluated minimizing or reversing the effects of fatty liver disease when mice fed high fat diets were used as controls (Dumas et al., 2006; Li et al., 2003; Lin et al., 2000; Xu et al., 2003).

Pup Mortality During Lactation and After Weaning A total of 117 pups in the F1.1 (first parity) generation were weaned on day 21, from which 40 pups were assigned dietary treatments and the remaining pups were placed on regular chow in the mouse colony. There was an unexpectedly high lactation and post weaning mortality rate in the F1.1 (first parity) generation. Overall, during the first pregnancy, pup mortality was 4.65% throughout lactation for dams fed the high fat) diet, and an 18% pup mortality for dams fed the low fat diet. Both of these mortality statistics exclude stillborn pups. Furthermore, a total of 20 pups died within four weeks post weaning. In the F1.2 (second parity) generation, a total of 38 pups died during lactation and 11 died post weaning out of a total of 155 pups born. Overall, in the second pregnancy, there was a 27% pup mortality rate through lactation when dams were fed the high fat diet, and a 20% pup mortality rate for dams fed the low fat diet, excluding still born pups. A few pups that were found dead and were recovered before mothers could eat them, were subjected to a necropsy evaluation. All three mice that were examined had at least one portion of the bowel that was necrotic (containing a black color). The small intestines of two out of the three mice were entirely necrotic and were covered in a sticky dark brown

to black liquid material. All three mice had soft, yellow colored bones, especially the ribs, which would indicate a calcium and phosphorus mineral deficiency. The unanticipated results from the average weekly weights and body composition, along with the abnormally high pup mortality rate during lactation and after weaning appeared to indicate that nutrient composition of our diets was inadequate. In order to test this theory, we observed a female mouse fed the HFr diet that gave birth to 7 pups. One pup died a day later, which is common if they have low viability. On day 21, which is the normal weaning day for healthy mouse offspring, 4 out of the remaining 6 pups were found dead by the university animal care staff and were removed from the cage. The 2 remaining alive pups were small, weak and were placed on heat. We checked on the mice an hour after the discovery of the dead pups and found 1 of the 2 remaining pups dead with the other pup looking very sickly. The remaining pup was humanely euthanized resulting in us not being able to wean any offspring from this dam. We suspected that milk production was significantly reduced in early lactation resulting in the pups being too immature at the time of weaning to survive. This female was removed from the study and fed our standard lab mouse chow (2018 from Teklad Envigo). She was then mated with one of the males for a third pregnancy. This dam subsequently gave birth to a litter of 9 pups, and on day 21 9 healthy normal weight pups were weaned with no mortality. This observation supported our diagnosis that the diets were deficient in an unknown nutrient.

Macronutrient and Mineral Analysis After macronutrient analysis, both the low fat and high fat diets contained adequate amounts of macronutrients (Table 6).

Table 7. Comparison of formulated and analyzed macronutrient composition of the low fat diet (LF), high fat diet (HF), and NRC (1995) requirements (mg/kg). Nutrient NRC (1995) LF LF HF HF requirement formulated analyzed formulated analyzed

(mg/kg) values (mg/kg) values values (mg/kg) (mg/kg) (mg/kg) Crude protein, % 18.0 18.3 17.0 18.2 17.4 Crude fat, % 5.0 3.1 3.3 11.6 11.5 Crude fiber, % 3.0 5.0 2.9 25.0 6.1 Carbohydrate1, % 50.0 67.2 64.4 39.1 57.3 1 Carbohydrate value calculated by: 100- (analytical results for moisture+ash+protein+fat) Since the macronutrients were present in adequate amounts in both the low fat and the high fat diet, we suspected that the diets were deficient in micronutrients. To confirm our suspicion of mineral deficiencies in these diets, we submitted samples of the low fat diets analysis. Results showed that both diets were severely deficient in all minerals (Table 7 and 8).

Table 8. Comparison of formulated and analyzed mineral composition of the low fat diet with NRC (1995) requirements (mg/kg dry matter). Element, NRC (1995) Formulated values Analyzed % Relative to mg/kg requirement values NRC (1995) requirement Ca 5,000 5,100 1,140 23% Cu 6 6 1.6 26% Fe 35 40 17 48% K 2,000 3,600 1,044 52% Mg 500 500 147 29% Mn 10 11 2.8 28% Na 500 1,400 294 59% P 3,000 3,200 986 33% Zn 10 35 9.4 94%

Table 9. Comparison of formulated and analyzed mineral composition of the high fat diet with NRC (1995) requirements (mg/kg dry matter). Element, NRC (1995) Formulated values Analyzed % Relative to mg/kg requirement values NRC (1995) requirement Ca 5,000 5,100 1,142 23% Cu 6,0 6.0 1.6 26% Fe 35 40 14 40% K 2,000 3,600 1,254 63% Mg 500 500 147 29% Mn 10 11 2.6 26% Na 500 1,400 805 161% P 3,000 3,200 1,001 33% Zn 10 35 9 92%

These startling results showed that both low fat and high fat diets were grossly deficiency in most of the required macro and micro minerals. This indicates that significant mineral deficiencies were the likely cause for the unexpected responses in body weights, body composition, and high mortality rates observed in this study. Trace mineral deficiencies of dams, especially during early gestation, have been shown to result in various malformations and abnormalities, and alter biochemical processes that are evident in adulthood (Carl L. Keen et al., 2003). Due to this finding, we were unable to test our original hypothesis nor interpret our results due to the confounding of feeding mineral deficient diets.

Conclusion The confounding effects of feeding mineral deficient diets prevented us from testing our original hypothesis. However, considering this fact, mice that were fed moderately high fat diets over a twelve-week period did not have body 61

weight, composition, and fatty liver responses similar to results previously reported in mice fed high fat diets containing at least 60% of calories from lipid. The physiological changes that were observed were opposite from the expected results, which could be due to feeding moderately high fat diets or trace mineral deficiencies of the diets. Therefore, it is still unknown if developmental programming is a result of maternal high fat diet or of maternal body composition.

Overall Summary Understanding the influences of maternal nutrition on developmental programming for the health of the offspring has recently been a popular focus of many researchers. By understanding the mechanisms of developmental programming, scientists can better recommend improved prenatal nutrition to mothers in an attempt to alleviate the increasing obesity rate in children. Studies have shown that maternal nutrition, especially when consuming a maternal high lipid diet, can negatively impact the development and later health and lifespan of offspring. However, all human and laboratory animal diets are formulated using Atwater system, which overestimates the energy values of feed ingredients. A more accurate system to use when formulating diets is the NE system due to its more comprehensive accounting for heat, fecal, urinary and methane losses of dietary components. The diets used in this study were formulated using the NE system, and were formulated to meet or exceed all of the mouse NRC requirements. The aim of this study was to determine if diet composition of maternal diets, independent of maternal body composition, would induce physiological changes in offspring. This was accomplished by feeding mice either a low fat restricted diet, high fat ad libitum diet, or high fat restricted calorie diet. We observed very unusual and unexpected results with no differences in body weights (dams and offspring), and mice fed the low fat diets had a greater percentage of body fat than mice fed the high fat diet. High pup mortality during lactation and post-weaning suggested that the nutrient composition of the

experimental diets was inadequate. After submitting diet samples for a complete nutrient analysis, we discovered that the diets were deficient in all minerals. Therefore, by feeding the mineral deficient diets, any effects of dietary energy responses were confounded, so the study needs to be repeated. Overall, maternal nutrition is crucially important for the health and lifespan of the offspring, especially mineral content.

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