For diets containing egg yolks or whites, hard- 

 boiled eggs were used. Yolks and whites were 

 separated, ground, and dried at 150° F. The 

 whites were reground after drying. The diet 

 containing a high level of egg yolk was reground 

 after mixing. 



Cholesterol or vitamin supplements, except for 

 choline, were added dry and mixed with the SPE 

 diet by blending in the Hobart mixer. Choline 

 was dissolved in water first and then added to the 

 dry ingredients before final blending. 



The diets were prepared fresh at least every 2 

 weeks and were kept refrigerated until used. All 

 of the fats and the ingredients containing fat 

 were kept under refrigeration. Aliquots of the 

 dietary ingredients and of the experimental diets 

 were kept for chemical analysis. 



The dietary ingredients and the experimental 

 diets were analyzed for moisture, nitrogen, fat, 

 and ash. Aloisture was determined by drying 

 in a vacuum oven at 70° C. Ash values were 

 obtained by incineration in a muffle furnace at 

 575° C. Nitrogen was determined by the 

 Kjeldahl-Wilfarth-Gunning method using mercury 

 as a catalyst (12) and distilling into boric acid 

 {175). Protein values were obtained by applying 

 to the nitrogen values the factors indicated. 

 Fat was determined by a modification 7 of the 

 AOAC acid hydrolysis procedure (18). Carbo- 

 hydrate values were calculated by subtracting 

 the weight of fat, protein, and ash from the total 

 dry weight. Mineral components were deter- 

 mined by spectrochemical analysis for the various 

 dietary ingredients and for most of the diets. 

 Those diets not actually analyzed were calculated 

 from the values for the dietary ingredients. 

 Gross calorie values were obtained for some of 

 the diets by use of the Parr Bomb Adiabatic 

 Calorimeter; others were calculated by application 

 of appropriate gross calorie values for the pi-oteins, 

 fats, and carbohydrates which they contained. 

 The values for thiamine, riboflavin, niacin, pyri- 

 doxine, folic acid, pantothenic acid, vitamin B ]2 , 

 choline, essential and nonessential amino acids, 

 fatty acids, and cholesterol were all calculated- 



Criteria for Evaluating Response to Diet 



Physical measurements, histological examina- 

 tion of the tissues, and limited chemical analyses 

 of liver, kidney, blood serum, and mine provided 

 the criteria used for evaluating the changes 

 occurring in the rat with age and/or with diet. 

 Measurements obtained on BHE rats fed the 

 stock diet provided information on the charac- 



7 Unpublished. 



teristics of the rats that served as a source of 

 the experimental animals. A group of rats fed 

 SP 8 HVO diet was included for comparative 

 purposes in each of the experimental series alinged 

 chiefly with modifications of this diet. Rats fed 

 SPE diet served as a basis of comparison for the 

 series of rats fed SPE diets containing various 

 supplements and for those fed high levels of egg 

 or egg fractions. 



Weight and intake data provided the informa- 

 tion needed to compare the rate of growth, 

 maximum weight attained, and efficiency of food 

 utilization. The influence of age and/or diet on 

 the size of the organs was determined by weighing 

 immediately, at the time of sacrifice, the liver, 

 right kidney, right adrenal, and right lobe of 

 the thyroid. 



Also, at the time of sacrifice, any gross abnor- 

 malities in the animals were noted. The left 

 kidney and adrenal, the left lobe of the thyroid, 

 and part of the median lobe of the liver (approxi- 

 mately 20 percent by weight of the total liver) 

 were fixed in 10 percent formalin to preserve these 

 tissues for histological examination. Other tissues 

 prepared routinely for histological examination 

 included the heart, lungs, aorta, salivary glands, 

 spleen, pancreas, and testes. These tissues were 

 embedded in paraffin, sectioned, and stained with 

 hematoxylin and eosin. 



The methods used for rating the microscopic 

 findings and the results of gross and microscopic 

 examination of the tissues are being reported in 

 detail elsewhere (54)- This report includes only 

 those phases of the histological findings that are 

 related to the chemical investigations reported, 

 and deals chiefly with the kidney and liver. 

 Damage to the liver and kidney was rated arbi- 

 trarily 1 to 4, with 4 the most extensive damage. 



Chemical analyses included determinations of 

 moisture, fat, protein, and ash in livers and kid- 

 neys', cholesterol and protein fractions in blood 

 serum, and coagulable protein in urine. Most of 

 the chemical analyses were obtained for rats that 

 were fasted before sacrifice. Immediately after 

 removal of the section of liver retained for histo- 

 logical examination, the weight of the remaining 

 portion was recorded and both the liver and kid- 

 ney were stored frozen, for chemical analysis. 

 Homogenates of these organs were prepared by 

 use of a Virtis homogenizer, and the various 

 analyses were carried out on weighed aliquots of 

 these homogenates. The values reported for the 

 liver content are for the whole liver, based on the 

 assumption that the composition of the sections 

 analyzed was similar to that of sections removed 

 for histological examination. 



During the early series, small kidneys were 

 pooled in order to obtain sufficient material for 



