A small group of young from a colony of Wistar 

 animals 5 raised on a different stock ration 6 was 

 also investigated to determine if the dietary 

 response would differ with another strain of rat. 

 To obtain information on the possible carryover 

 effect of the diet of the parents, a few BHE rats 

 were raised on the stock diet on which the parents 

 of the Wistar rats had been maintained and the 

 response of their young to diet was determined. 

 Male rats were used throughout these investiga- 

 tions because preliminary studies had indicated 

 that males were more susceptible than females to 

 certain dietary regimens that caused early death 

 and accelerated degenerative changes in the 

 tissues. 



Rats were placed on the experimental diets at 

 weaning, when they were between 21 and 24 days 

 of age. The majority of weanling rats weighed 

 between 40 and 50 grams. Except for one series, 

 the animals were maintained on a constant diet 

 throughout life. To determine whether the re- 

 sponse to diet could be influenced by the age at 

 which consumption of the diet was begun, the 

 influence of changing the dietary regimen at 250 

 days of age was investigated. For each series, 

 only those litters were used that contained enough 

 males to permit placing one littermate on each 

 experimental diet in the series. The average 

 initial weights of the animals fed each diet were 

 kept as nearly uniform as possible. 



All animals were kept in an air-conditioned 

 laboratory maintained at a temperature of 78°- 

 82° F. and at a relative humidity which averaged 

 37 percent, although not rigorously controlled. 

 The animals were fed ad libitum and had access to 

 water at all times. The animals fed the pelleted 

 stock ration were taken directly from the stock 

 colony. They were housed five to six to a cage, 

 and no data were obtained on their food intake. 

 Rats fed the experimental diets were housed in 

 individual metal cages with raised screen floors. 

 When urine collections were made, the animals 

 were transferred to wire metabolism cages with 

 half-inch mesh bottoms supported above glass 

 funnels 9 inches in diameter. 



The rats fed experimental diets were weighed 

 daily for the first 2 to 3 weeks and weekly there- 

 after. For many of the series, food intake 

 records were maintained. Food intake was 

 recorded daily during the first 2 to 3 weeks 

 and twice weekly throughout the remainder of 

 their life. Scattered food was collected and 

 weighed at the time of recording food intake. 

 Observations were made regularly of the general 

 physical condition of the annuals. 



5 Kindly supplied to us by Arthur M. Hartman, Animal 

 Husbandry Research Division, Agricultural Research 

 Service, Beltsville, Md. 



6 Stock colony ration consisted of: yellow cornmeal, 

 68.5 percent; linseed oil meal, 14.0 percent; meat scrap, 

 9.0 percent; commercial casein, 4.0 percent; alfalfa meal, 

 2.0 percent; bone meal, 2.0 percent; and sodium chloride, 

 0.5 percent. Supplements of lettuce and carrots were fed 

 once a week. 



For some experiments, urine was collected over 

 a 7-hour period, during which time the animals 

 had access to water but not to food; for others, 

 urine was collected over a 16- to 17-hour period, 

 with the rats having access to both food and water. 

 To collect the urine samples, 50-ml. glass centrifuge 

 tubes containing toluene were placed under glass 

 funnels. Plugs of extremely fine wire mesh were 

 placed in the neck of each funnel to screen out 

 feces, feed, and hair. 



To determine the changes that occur with 

 aging, some animals were sacrificed at scheduled 

 ages while they were maintaining or gaining 

 weight and were showing no obvious signs of 

 illness. Other animals designated for survival 

 studies were continued on the experimental diets 

 until they became obviously ill. These rats 

 reduced their food intake or stopped eating 

 entirely, they became listless, and their coats 

 became rough. These animals consistently lost 

 weight and occasionally suffered from obvious 

 respiratory disturbances. 



It was not possible, without 24-hour vigilance, 

 to keep animals until they died naturally and 

 still obtain tissues suitable for microscopic exami- 

 nation. Thus the results of the longevity studies 

 were dependent upon arbitrary decisions as to 

 when the rats were approaching death. At first, 

 animals were allowed to continue until extremely 

 ill, with weight losses often equal to as much as 

 one-third of their maximum body weight. Un- 

 expected deaths resulted in failure to obtain 

 tissues that were suitable for histologic or chemical 

 analysis because of post-mortem changes. Later, 

 to avoid loss of suitable tissues, rats for longevity 

 studies were sacrificed as soon as there was con- 

 sistent weight loss for at least 3 weeks and/or 

 when weight loss exceeded 50 grams. 



During the early investigations, rats were sacri- 

 ficed by injecting 0.5 ml. of 2 percent solution of 

 sodium amytal per 100 grams body weight without 

 a preliminary fasting period. When the criteria 

 under investigation were extended to include 

 measurements of various serum protein fractions, 

 it became necessary to sacrifice the animals after 

 a 17-hour fast and to obtain blood samples by 

 cardiac puncture. 



Diets 



The semipurified diet, which served as a basis 

 for most of the experimental diets, consisted of: 



Ingredient Percent 



Casein " 16 



Lactalbumin 2 8 



Yeast 3 10 



Salt mixture 4 4 



Hydrogenated vegetable oil (HVO) 5 8 



Sucrose 52 



Celluflour 2 



i High-nitrogen, acid-washed, edible product, from Sheffield Farms Co. 



2 Labeo Lactalbumin containing some lactose, from Borden Co. 



s Dried brewer's yeast, type 200B, from Standard Brands, Inc. 



< Source: Osbome and Mendel (149). 



« Crisco, from Procter & Gamble Co., Cincinnati, Ohio. 



