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vi CHAPTER 1: GENERAL INTRODUCTION 1 Egg Nutrition 1 Rational for Nutrient Enrichment 2 Rational for Distribution Determination 5 Thesis Organization 5 References 6 CHAPTER 2: LITERATURE REVIEW 9 Vitamin E: Tocopherols and Tocotrienols 9 Absorption and Transport Vitamin E 12 Annatto Tocotrienols 14 Cholesterol Lowering 15 Cholesterol Quantification 18 Egg Formation 19 References 22 CHAPTER 3: SUPPLEMENTATION OF LAYING-HEN FEED WITH ANNATTO TOCOTRIENOLS AND IMPACT OF α-TOCOPHEROL ON TOCOTRIENOL TRANSFER TO EGG YOLK 27 Abstract 28 Introduction 29 Materials and Methods 30 Results and Discussion 36 Conclusions 46 References 48 Tables and Figures 5428 Introduction 29 Materials and Methods 30 Results and Discussion 36 Conclusions 46 References 48 Tables and Figures 54

Cholesterol, in free or esterified form, is a sterol that is found in animal products. Free cholesterol is one of the unsaponifiable matters, so most quantification methods involve saponification of the acyl lipids under heating and alkaline conditions to obtain the unsaponifiable fraction for further GC quantification. We examined the stability of cholesterol in the harsh saponification environment and the completeness of the hydrolysis reaction of cholesterol ester. Cholesterol was found to be stable in a model saponification system. However, only about 50 % of the cholesterol ester was hydrolyzed with a long saponification time (up to 18 h). This incomplete reaction will lead to an underestimation of the total cholesterol if a sample has a significant amount of sterol esters. In this study, 5.1 % of the total sterol in egg yolk was found to be esterified cholesterol, but yolk’s total cholesterol content quantified by using standard methods was lower than that determined using a commercial kit that measures total cholesterol. More research is needed to further understand the stability and reactivity of both free and esterified cholesterol in foods in order to use appropriate methodology for cholesterol quantification.

In this 60-wk study, egg quality, egg shelf-life, egg cholesterol content, total yolk lipids, and yolk fatty acid composition of eggs produced by Dekalb white laying hens in commercial aviary houses with either light-emitting diode (LED) or fluorescent (FL) lighting were compared. All parameters were measured at 27, 40, and 60 wk of age, except for egg shelf-life, which was compared at 50 wk of age. The results showed that, compared to the FL regimen, the LED regimen resulted in higher egg weight, albumen height, and albumen weight at 27 wk of age, thicker shells at 40 wk of age, but lower egg weight at 60 wk of age. Egg quality change was similar between the lighting regimens during the 62-d egg storage study, indicating that LED lighting did not influence egg shelf-life. Eggs from both lighting regimens had similar cholesterol content. However, cholesterol concentration of the yolk (15.9 to 21.0 mg cholesterol/g wet weight yolk) observed in this study was higher than that of United States Department of Agriculture (USDA) database (10.85 mg/g). No significant differences in total lipids or fatty acid composition of the yolks were detected between the two lighting regimens.

The impact of supplementing laying-hen feed with annatto tocotrienols (T3s) and alpha-tocopherol on the distribution of various forms of vitamin E and cholesterol throughout the hen's body was evaluated. A total of 18 organs or tissues (skin, fat pad, liver and gall bladder, heart, oviduct, forming yolk, laid yolk, lungs, spleen, kidney, pancreas, gizzard, digestive tract, brain, thigh, breast, manure, and blood) were collected after 7 wk of feeding on diets enriched with various levels of alpha-tocopherol and annatto extract that contained gamma-T3 and delta-T3. Tissue weights, contents of lipid, alpha-tocopherol, gamma-T3, delta-T3, cholesterol, and fatty acid composition of extracted lipids from the collected organs and tissues were determined. Tissue weight and lipid content did not change significantly with feed supplementation treatments, except that the liver became heavier with increased levels of supplementation. Overall, the main organs that accumulated the supplemented vitamin E were fat pad, liver and gall bladder, oviduct, forming yolks, laid yolks, kidney, brain, thigh, and breast. Much of annatto gamma-T3 and delta-T3 (> 90%) was found in the manure, indicating poor uptake. In some tissues (brain and oviduct,) a significant increase in polyunsaturated fatty acids was seen with increased supplementation. Alpha-tocopherol impacted the transfer of gamma-T3 to forming and laid yolks, but did not impact delta-T3 transfer. No significant differences were found in most of the tissues in cholesterol, except a reduction in heart, based on tissue as-is. Blood samples showed large variations in individual hens with no significant differences in total and HDL cholesterol, or total triacylglycerols. Supplementing feed with annatto T3s and alpha-tocopherol showed that the vitamin E profile and distribution of the laying-hen body can be altered, but to different extents depending on tissue. The result of this research has significance in enhancing meat nutrient content.

Hens can efficiently transfer nutrients from their feed to the eggs. Tocotrienols (T3s) have various health benefits including lowering cholesterol. Annatto is the only known source of T3s without the presence of α-tocopherol; hence it can be used to study T3 transfer without the interference of α-tocopherol. In this study, hens were fed diets for 7 weeks containing annatto at 100, 500, or 2000 ppm (by weight) and also 2000 ppm annatto with 200, 600, or 1000 ppm of added α-tocopherol to study the effect of α-tocopherol on transfer of T3s. No significant differences were found in egg production or properties. Significant differences (p < 0.05) were found in transfer efficiencies of tocopherol and T3s to the yolks. α-Tocopherol was transferred more efficiently (21.19-49.17%) than γ-T3 (0.50-0.96%) or δ-T3 (0.74-0.93%). Addition of 1000 ppm of α-tocopherol decreased the amount of γ-T3 but did not impact the transfer of δ-T3 to the egg. These feeding treatments did not impact the cholesterol content of the eggs.