Incorporating the best findings from current, high-quality research into routine clinical practice is the basis of evidence-based care. Chapter 1: "Systematic Review and Meta-Analysis in Evidence-Based Care" is a review of the systematic review process, including meta-analysis, aimed at clinical professionals with limited statistical training. It advocates the use of the systematic review process, outlines some general techniques, and provides selected resources where individuals can acquire additional assistance. The typical steps involved include: formulating a clear research question, defining inclusion and exclusion criteria, extracting the data and assessing the study quality, summarizing and synthesizing the evidence, and then interpreting the findings. When effort is made to minimize bias and locate as many articles on a particular topic as possible, systematic reviews and meta-analyses can produce invaluable findings for evidence-based care. Chapter 2: "The Effect of Macronutrient Distribution on the Lipid Profile in Adults: A Systematic Review and Meta-Analysis" describes a systematic review and meta-analysis that examined the impact total macronutrients had on blood lipid levels. This chapter builds upon the concepts introduced in chapter one, and assesses the effect of manipulating macronutrient distribution on the lipid profile of adults, and compares these effects to recommendations regarding macronutrients, such as the Acceptable Macronutrient Distribution Ranges (AMDRs). Suggestions related to improving the quality of meta-analyses are also outlined, and supplemental analyses are provided at the end of the dissertation.

In setting the AMDR for protein at 10-35% of daily energy, the Institute of Medicine acknowledged a lack of data regarding the safety of long-term intakes. The current study assessed the impact of chronic (17 months) protein consumption at the upper end of the AMDR on renal function, histology, and inflammation. Using plant and animal whole protein sources, female Sprague-Dawley rats (70 days old; n=8-11 at 4, 8, 12, or 17 mo.) were randomized to either a normal (NP; 15% of energy) or high protein (HP; 35% of energy) diet. Egg albumen and skim milk replaced carbohydrates in the HP diet. Diets were balanced for energy, fat, vitamins and minerals, and offered ad libitum. Renal function was analyzed by creatinine clearance and urinary protein levels. Glomerular hypertrophy, glomerulosclerosis and tubulointerstitial fibrosis were assessed on kidney sections. Kidney disease progression was determined by the measurement of transforming growth factor beta-1 (TGF-β1) and renal inflammation by the measurement of chemokines monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation normal T-cell expressed and secreted (RANTES). Rats consuming the HP compared to NP diet had ~17% higher kidney weights (P<0.0001) and ~4.8 times higher proteinuria (P<0.0001). There was a trend towards higher creatinine clearance with HP (P=0.055). Consistent with this, HP compared to NP rats had ~22% larger glomeruli (P<0.0001) and ~33% more glomerulosclerosis (P=0.0003). The HP diet had no significant effect on tubulointerstitial fibrosis and renal TGF-β1 levels and did not result in higher renal levels of MCP-1 and RANTES. In fact, per mg renal protein, HP rats had ~16% lower MCP-1 (P<0.0001) and ~34% lower levels of RANTES (P<0.0001) than NP. The absence of an increase in cytokine levels may be a reflection of the moderate changes in renal pathology observed in rats offered HP diets. These data in normal female rats suggest that protein intakes at the upper end of the AMDR are detrimental to kidney health in the long-term. While modest, this may have implications for individuals whose kidney function is compromised, especially given the prevalence of those unaware of their kidney disease within North America.

In setting the AMDR for protein at 10-35% of daily energy, the Institute of Medicine acknowledged a lack of data regarding the safety of long-term intakes. The current study assessed the impact of chronic (17 months) protein consumption at the upper end of the AMDR on renal function, histology, and inflammation. Using plant and animal whole protein sources, female Sprague-Dawley rats (70 days old; n=8-11 at 4, 8, 12, or 17 mo.) were randomized to either a normal (NP; 15% of energy) or high protein (HP; 35% of energy) diet. Egg albumen and skim milk replaced carbohydrates in the HP diet. Diets were balanced for energy, fat, vitamins and minerals, and offered ad libitum. Renal function was analyzed by creatinine clearance and urinary protein levels. Glomerular hypertrophy, glomerulosclerosis and tubulointerstitial fibrosis were assessed on kidney sections. Kidney disease progression was determined by the measurement of transforming growth factor beta-1 (TGF-β1) and renal inflammation by the measurement of chemokines monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation normal T-cell expressed and secreted (RANTES). Rats consuming the HP compared to NP diet had ~17% higher kidney weights (P<0.0001) and ~4.8 times higher proteinuria (P<0.0001). There was a trend towards higher creatinine clearance with HP (P=0.055). Consistent with this, HP compared to NP rats had ~22% larger glomeruli (P<0.0001) and ~33% more glomerulosclerosis (P=0.0003). The HP diet had no significant effect on tubulointerstitial fibrosis and renal TGF-β1 levels and did not result in higher renal levels of MCP-1 and RANTES. In fact, per mg renal protein, HP rats had ~16% lower MCP-1 (P<0.0001) and ~34% lower levels of RANTES (P<0.0001) than NP. The absence of an increase in cytokine levels may be a reflection of the moderate changes in renal pathology observed in rats offered HP diets. These data in normal female rats suggest that protein intakes at the upper end of the AMDR are detrimental to kidney health in the long-term. While modest, this may have implications for individuals whose kidney function is compromised, especially given the prevalence of those unaware of their kidney disease within North America.

Substantial evidence supports a role of diet in glucose metabolism, but only a few reports have investigated the impact of diet during pregnancy on risk of gestational diabetes (GDM). Although metabolic hormones have been detected in milk, no studies have investigated the impact of maternal metabolic status assessed during pregnancy on insulin and adiponectin concentrations in human milk. The purpose of this thesis was to investigate the association of maternal macronutrient intakes with metabolic status during pregnancy and its subsequent impact on human milk hormones. Participating women (n=216) underwent a 3-hour oral glucose tolerance test at 30 (95% confidence interval [CI] 25, 33) weeks gestation, recalled their second trimester dietary intake, and donated early (the first week) and mature (3 months postpartum) milk. Higher vegetable and fruit fiber intake was associated with reduced insulin resistance (beta±SE -0.100±0.029, p=0.0008) and increased insulin sensitivity (0.029±0.012, p=0.01) among those with a family history of type 2 diabetes. Lower % carbohydrate and higher % total fat were associated with increased GDM risk (odds ratio 0.60 [95% CI 0.40, 0.90] and 1.61 [1.06, 2.44], respectively). Prenatal metabolic abnormalities including higher pregravid body mass index (beta±SE 0.053±0.014, p=0.0003), in addition to gravid hyperglycemia (0.218±0.087, p=0.01), insulin resistance (0.255±0.047, p<0.0001), lower insulin sensitivity (-0.521±0.108, p<0.0001), and higher serum adiponectin (0.116±0.029, p<0.0001) were associated with higher insulin in mature milk. Obstetrical measures including nulliparity (0.171±0.058, p=0.004), longer duration of gestation (0.546±0.146, p=0.0002), and unscheduled caesarean section (0.387±0.162, p=0.02) were associated with higher adiponectin in early milk. Holder pasteurization, a process recommended by the Human Milk Bank Association of North America before distributing human donor milk, reduced milk adiponectin and insulin concentrations by 32.8% and 46.1%, respectively (both p<0.0001). In conclusion, the distribution of macronutrient intakes during pregnancy was associated with risk for abnormal glucose metabolism later in pregnancy. In addition, maternal prenatal metabolic abnormalities were associated with high insulin concentrations in mature milk, while only obstetrical parameters were associated adiponectin concentrations in early milk. Our findings support the need for continued work to determine optimal prenatal nutritional strategies to prevent GDM and subsequently to improve infant nutrition.

Substantial evidence supports a role of diet in glucose metabolism, but only a few reports have investigated the impact of diet during pregnancy on risk of gestational diabetes (GDM). Although metabolic hormones have been detected in milk, no studies have investigated the impact of maternal metabolic status assessed during pregnancy on insulin and adiponectin concentrations in human milk. The purpose of this thesis was to investigate the association of maternal macronutrient intakes with metabolic status during pregnancy and its subsequent impact on human milk hormones. Participating women (n=216) underwent a 3-hour oral glucose tolerance test at 30 (95% confidence interval [CI] 25, 33) weeks gestation, recalled their second trimester dietary intake, and donated early (the first week) and mature (3 months postpartum) milk. Higher vegetable and fruit fiber intake was associated with reduced insulin resistance (beta±SE -0.100±0.029, p=0.0008) and increased insulin sensitivity (0.029±0.012, p=0.01) among those with a family history of type 2 diabetes. Lower % carbohydrate and higher % total fat were associated with increased GDM risk (odds ratio 0.60 [95% CI 0.40, 0.90] and 1.61 [1.06, 2.44], respectively). Prenatal metabolic abnormalities including higher pregravid body mass index (beta±SE 0.053±0.014, p=0.0003), in addition to gravid hyperglycemia (0.218±0.087, p=0.01), insulin resistance (0.255±0.047, p<0.0001), lower insulin sensitivity (-0.521±0.108, p<0.0001), and higher serum adiponectin (0.116±0.029, p<0.0001) were associated with higher insulin in mature milk. Obstetrical measures including nulliparity (0.171±0.058, p=0.004), longer duration of gestation (0.546±0.146, p=0.0002), and unscheduled caesarean section (0.387±0.162, p=0.02) were associated with higher adiponectin in early milk. Holder pasteurization, a process recommended by the Human Milk Bank Association of North America before distributing human donor milk, reduced milk adiponectin and insulin concentrations by 32.8% and 46.1%, respectively (both p<0.0001). In conclusion, the distribution of macronutrient intakes during pregnancy was associated with risk for abnormal glucose metabolism later in pregnancy. In addition, maternal prenatal metabolic abnormalities were associated with high insulin concentrations in mature milk, while only obstetrical parameters were associated adiponectin concentrations in early milk. Our findings support the need for continued work to determine optimal prenatal nutritional strategies to prevent GDM and subsequently to improve infant nutrition.

To help mitigate the escalating prevalence of Type 2 Diabetes (T2D) and alleviate society of its associated morbidity and economic burden on health care, it is crucial to understand its aetiology. Both genetic and the environmental risk factors are known to be involved. Healthy diets have been proven to reduce the risk of T2D in primary prevention trials, however, which components and exact mechanisms are involved is not fully understood, in particular, the role of macronutrient intake. Body weight, glycaemic markers and T2D are all to some extent genetically regulated. There may also be genetic influences on how people digest, absorb or metabolise macronutrients. This poses the possibility that the interplay between genes and our diet may help us unravel T2D’s aetiology. The aim of this PhD was to investigate gene-diet interactions on the risk of incident T2D, focusing primarily on macronutrient intake as the dietary factor. First, I systematically evaluated the current evidence before taking a step-wise approach (hypothesis driven to hypothesis-free) to interrogate gene-macronutrient interactions. This identified 13 publications, with 8 unique interactions reported between macronutrients (carbohydrate, fat, saturated fat, dietary fibre, and glycaemic load derived from self-report of dietary intake and circulating n-3 polyunsaturated fatty acids) and genetic variants in or near TCF7L2, GIPR, CAV2 and PEPD (p < 0.05) on T2D. All studies were observational with moderate to serious risk of bias and limitations that included lack of adequate adjustment for confounders, lack of reported replication and insufficient correction for multiple testing. Second, these reported interactions did not replicate in a large European multi-centre prospective T2D case-cohort study called EPIC-InterAct. We concluded that the heterogeneity between our results and those published could be explained by methodological differences in dietary measurement, population under study, study design and analysis but also by the possibility of spurious interactions. Third, given the paucity of gene-macronutrient interaction research using genetic risk scores (GRS), we examined the interaction between three GRS (for BMI (97 SNPs), insulin resistance (53 SNPs) and T2D (48 SNPs)) and macronutrient intake (quantity and quality indicators) in EPIC-InterAct. We did not identify any statistically significant interactions that passed multiple testing corrections (p≥0.20, with a p value threshold for rejecting the null hypothesis of 0.0015 (based on 0.05/33 tests)). We also examined 15 foods and beverages identified as being associated with T2D, and no significant interactions were detected. Lastly, we applied a hypothesis-free method to examine gene-macronutrient interactions and T2D risk by using a genome-environment-wide-interaction-study. Preliminary findings showed no significant interactions for total carbohydrate, protein, saturated fat, polyunsaturated fat and cereal fibre intake on T2D. In conclusion, the consistently null findings in this thesis using a range of statistical approaches to examine interactions between genetic variants and macronutrient intake on the risk of developing T2D have two key implications. One, based on the specific interactions examined, this research does not confirm evidence for gene-diet interactions in the aetiology of T2D and two, this research suggests that the association between macronutrient intake and the risk of developing T2D does not differ by genotype.

Ethiopia maintains an extensive livestock population; however, reproductive performance of cattle and their breeding management are unsatisfactory. Currently, the sole diagnostic tool in the country is rectal palpation, which is inaccurate for early pregnancy in cattle. The study assessed reproductive performance and major reproductive problems using questionnaire survey, and evaluated simple, cost-effective alternative monitoring approaches using on-farm diagnostic tools to determine milk and serum progesterone (P4) and evaluate reproductive status. There were 177 dairy farms (range 1-115 cattle per farm) included in the questionnaire survey. Of these, 47 participated in the quantitative determination of P4 and estradiol profiles that used an enzyme-linked immunosorbent assay (ELISA) and in the on-farm diagnostic trial that used qualitative ELISA (Target P4 and Dipstick (P4 Rapid), and the reproductive status of 319 crossbred [Holstein Friesian (HF) X Zebu] dairy cattle was assessed. Questionnaires indicated that heifers in the study area reach puberty at older age and calve late, and cows have long postpartum estrus and calving interval. Anestrus, repeat-breeding, dystocia, retained fetal membranes (RFM), endometritis and abortion as the major reproductive problems in dairy cattle in the study area. Serum, milk, saliva and urine P4 and serum estradiol profiles of cattle at different reproductive status was determined using quantitative laboratory ELISA. High P4 levels was detected in pregnant and diestrus cattle than cattle that were anestrus and in-heat. Estradiol level was higher in cattle that were in-heat than cattle in other reproductive conditions. On-farm P4 ELISA indicated in-heat (estrus) 10 (3.1%), anestrus 77 (24.2%), repeater (follicular cyst) 9 (2.8%), normally cycling 69 (21.6%) and pregnant 154 (48.3%). The field P4 ELISA findings were validated using quantitative laboratory P4 ELISA, and similar results were obtained. The sensitivity and specificity of on-farm and laboratory P4 ELISA tests for diagnosing pregnancy were 88.6 & 99.4% and 98.1 & 100%, respectively. Once reproductive problems were identified using on-farm P4 ELISA and per rectal palpation, along with reproductive history, 122 cattle (75 cows and 47 heifers) were assigned to a 10-day Controlled Internal Drug Release (CIDR) in combination with prostaglandin F2-alpha (PGF2α) and equine chorionic gonadotrophin (eCG) based estrus synchronisation protocol to study the estrus response and conception rate. The overall estrus response and conception rates were 97.5% and 78.3%, respectively, with no significant differences in parity, pre-treatment reproductive status (anestrus or cycling (repeat-breeding/silent-estrus)) and farming system (smallholder vs organised commercial farms). The study has shown high estrus response and conception rate. Hence, this protocol is highly recommended to enhance fertility of dairy cattle in the study area and other regions. Finally, the study has determined the macronutrient composition of milk and assessed the effect of reproductive status, farm (nutritional) management, stages of lactation, parity and breed on milk composition in 246 dairy cows. This consisted of 184 crossbred cows from smallholder (n= 76: 36 non-pregnant and 40 pregnant) and organised commercial dairy farms (n= 108: 62 non-pregnant and 46 pregnant) in and around Mekelle, Ethiopia and by way of comparison, 62 HF cows (25 non-pregnant and 37 pregnant), either managed indoors or outdoors in a commercial farm in Edinburgh, United Kingdom. The mean milk fat, protein, lactose, total solid (TS) and solid-no-fat (SNF) recorded were 2.36%, 3.46%, 4.37%, 10.39% and 7.82%, in crossbred dairy cows, compared with 5.05%, 3.71%, 4.72%, 13.68% and 8.43%, in HF cows, respectively. Significantly lower (p < 0.05) level of macronutrient was recorded in milk from crossbred cows than HF cows. Milk fat in both breeds was affected by reproductive status, farm (nutritional) management and stages of lactation, but not by parity. In both cattle breeds, the milk fat content was significantly higher (p < 0.05) in pregnant than non-pregnant cows. Milk protein content was significantly (p < 0.05) affected by reproductive status (in crossbreds) and stages of lactation (in both breeds), but not by farm management or parity. Reproductive status (in crossbreds), stages of lactation (in both breeds) and parity (in crossbreds) affected lactose level; however, farm management had no effect on lactose level in both breeds. Milk fat was the most affected macronutrient content in both breeds. Low milk fat level in crossbred cows could be as a result of samples obtained from early milking coupled with nutritional management. In conclusion, the present study has determined the major reproductive problems in crossbred dairy cattle, assessed their actual reproductive status using rapid, cost effective, simple and applicable on-farm P4 tests, and established P4 and estradiol profiles at different reproductive status. The major breeding problem was poor estrus detection evidenced when animals reported anestrus were confirmed normally cycling using on-farm as well as laboratory P4 assays. These studies offer opportunities for establishing simple field reporting of reproductive status in these crossbred dairy cattle, which can have a major impact on breeding management and productivity.

Dietary restriction (DR), is a reduction in food intake, either through overall calorie or specific macronutrient intake, while avoiding malnutrition. DR has been consistently shown to increase longevity and protect against age related diseases. Although originally thought to be the result of a reduction in caloric intake, recent evidence suggests that the ratio of macronutrients, particularly that of protein : non-protein energy, also plays a role. The broad range of species in which DR is known to be effective, suggests an evolutionary conserved mechanism. However, the suggestion of a strong model species bias and a potential sex bias have led some to question the ubiquity of responses to DR. Here, I address the following questions: (i) How consistent is the effect of DR on reproduction? (ii) How does varying macronutrient intake effect both growth and body composition in three-spine sticklebacks (Gasterosteus aculeatus)? (iii) What is the effect of dietary macronutrient intake on lifespan and reproduction in the three-spine stickleback? and (iv) How does changing macronutrient intake impact fitness related traits, such as condition and performance in three-spine sticklebacks? Through use of a systematic review and meta-analysis, I show that the effect of DR on reproduction is evolutionarily conserved, though the effect is stronger in model species. However, when accounting for all significant moderators there is no evidence of the suggested sex differences in the effect of DR. I show that body composition is predicted by dietary lipid intake, with sticklebacks targeting a lower ratio of protein : fat within the body, potentially via metabolism and excretion of protein. These results hint at a link between conversion and excretion of protein and survival costs associated with high protein diets. I show that mortality risk is reduced at balanced protein : lipid intakes in males and generally at low protein : lipid intakes for females. However, the effect in females is not consistent throughout life. I further show that reproduction is maximised on high protein : lipid intakes for both sexes. These results suggest a macronutrient mediated trade-off between lifespan and reproduction in male three-spine sticklebacks. Finally I show a positive effect of lipid intake on male condition (a possible indicator of overall health) hinting at a relationship between lipid intake, adiposity, health and lifespan in male three-spine sticklebacks.

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