mm, RHH' &' \ : ■■ r mv$:m The Growth of the Body and Organs in Albino Bats Fed With a Lipoid-Free Bation y -'T‘V •• , rV-- y$U ' { . • . SHINKISHI HATAI The Wistar Institute of Anatomy and Biology Reprinted from The Anatomical Record, Vol. 9, No. 1, January, 1915 ' Ay ■ if&'AiY' ’ . V,; ; ' .-4 ;< i-itt ’ A-- . ■. . * V'-i v ‘ >''S \ >i ^=5-3 * Reprinted from The Anatomical Record, Vol. 9, No. 1 January, 1915 THE GROWTH OF THE BODY AND ORGANS IN ALBINO RATS FED WITH A LIPOID-FREE RATION Nl «r> CM ly I >■» i 3£ SHINKISHI HATAI The Wistar Institute of Anatomy and Biology Nearly seven years ago the writer attempted raise stunted albino rats with the hope that a forced retardation of growth would induce some disturbance in the firm relation whick-dfbr- mally exists between the weight of the body and of the oentral nervous system. The stunted rats were produced by feeding them with a minimum amount of nitrogenous food. It was found, however, that in this instance the artificial stunting did not modify the weight relation between the body and the central nervous system (Hatai ’08). Although it was highly desirable to pursue this investigation further, yet on account of incon¬ stancy and uncertainty of the outcome in raising stunted rats the method employed, the investigation was postponed. 1911 Professors Osborne and Mendel published a series of remarkable papers in which the results of maintenance experi¬ ments by means of various isolated proteins were fully described. According to these investigators, albino rats about one-third grown can maintain their body weight for a considerable period without revealing any sign of nutritional or physical deterior¬ ation. This satisfactory and constant procedure for producing undersized rats renewed my interest in the problem mentioned. During the past two years I have been so fortunate as to receive a number of stunted rats with their controls for exami¬ nation. These came through the courtesy of Dr. McCollum, who raised the rats by feeding them with a 'lipoid-free ration.’ These rats fall into two series: the series of 1913 and the series of 1914. The present paper contains the results of the ana¬ tomical examination of these interesting rats, and I take this THE anatomical RECORD, VOL. 9, NO. 1 JANUARY, 1915 REMC ? / 2 SHINKISHI HATAI opportunity to thank Dr. McCollum for his courtesy in putting these animals at my disposal. The rats used were from those bred in the colony at The Wistar Institute in Philadelphia and sent to the University of Wisconsin. In each case rats belonging to the same litter were divided by Dr. McCollum into two lots with nearly identical body weights. The one lot was used for control and received the normal mixed ration, while the other lot, which was used for the experiment, received a specially prepared diet. As to the dietary formula, the following statements were kindly furnished by Dr. McCollum: The ration of the experimented rats which received the lipoid-free food was as follows: Casein . 18 per cent Agar-agar . 2 per cent Lactose . 20 per cent Dextrin . 56 per cent The salts were as stated below: Per 100 grams of ration Salt mixture No. 17 4 No. 185 gm. gm. NaCl . 0.808 0.168 MgS04 (anhydrous) . 0.264 0.264 NaH2P04H20 . 0.336 0.336 K2HP04 . 0.936 0.964 CaH4(P0)2H20 . 0.528 0.528 Fe citrate . 0.096 0.096 Ca lactate . 2.000 1.300 The salt mixtures no. 174 and no. 185 were given at different periods in the case of both series. At the end of the experiment these rats were shipped back to The Wistar Institute for the anatomical examination, where the writer determined the weights of the following organs: Brain and spinal cord, heart, lungs, kidneys, liver, spleen, alimentary tract, testes and ovaries, suprarenals, thymus, thyroid, hypophy¬ sis and eyeballs. Some of these organs were preserved for fur¬ ther histological examination. Besides the organs mentioned, the bones also were examined. Although the methods employed in determining the relative amount of alteration in the various organs of the experimented rats, and also the technique for the preparation of the bones and separation of the encephalon into the four parts can be found GROWTH OF BODY AND ORGANS 3 in my papers recently published (Hatai ’13 and ’14), I shall briefly restate the essential points. The encephalon was divided into four parts in the following way: 1. Olfactory bulbs. The protruding portions of the olfactory tract with bulbs were cut from the rest of the encephalon by section of the tract just caudad to the bulb. 2. Cerebrum. The cerebrum is separated from the stem by a cut passing just in front of the dorsal edge of the anterior col¬ liculi and just caudad to the corpus mammillare on the ventral surface. 3. Cerebellum. The cerebellum is separated by severing the peduncles. 4. Stem. The structure which is left after removal of these three parts mentioned above, is called the stem. The bones were prepared as follows: The bones are freed from the main bulk of muscles and placed in a hot aqueous solution of 2 per cent 'gold dust washing powder.’ After macer¬ ation for several hours at nearly 90°C., the remaining soft parts are removed. The bones thus prepared are gently wiped with blotting paper and are weighed. This gives the 'fresh weight.’ These weighed bones are then dried at 95°C. for one week and the amount of moisture determined from the weight of the dried residue. In order to determine the amount of modification following the experimental ration, we have employed our usual method of comparing the observed values with those found in a series of reference tables that have been compiled in this laboratory. These tables present for normal rats adequate data on all the organs and characters under consideration and in each case the graph representing the table can be expressed by a mathemat¬ ical formula (Hatai T3; Hatai T4). In making the comparison between the observed values and those in the tables — the body length is always used as the basal measurement and the weight of the body or organs as observed compared with the corresponding values given in the reference tables. In this manner comparison is made not only for the 4 SHINKISHI HATAI experimented rats but also for those used as controls. The de¬ partures of the observed values from those in the tables having been observed in each case — the difference between that found for the experimented animals and that for the controls is ob¬ tained and this figure is used to indicate the amount by which the experimented animals have been modified. Two examples will serve to illustrate this procedure. They are taken from table 3 — C, normal males, 1914 series: (1) On the ‘mixed ration’ the average tail length for the three rats is 172 mm., for the given body length, 196 mm. We expect from the reference tables a tail length of 165 mm. The observed value is therefore plus 4.2 per cent. The two rats on the “lipoid-free diet and egg fat” give a tail length of 151 mm. for a body length of 168 mm. From the reference tables we should expect a tail length of 139 mm. The observed value for the tail length of the experimented group is therefore plus 8.6 per cent. The difference between these two percentage shows the tail length in the experimented group to be 8.6 — 4.2, or 4.4 per cent greater than that of the controls. This is the value given in table 3. (2) Taking the brain weights for the groups just used we find by following the method employed above for the tail length, that the group on the “mixed ration” has a brain weight 4.8 per cent below the reference table value, while the group on the “lipoid-free ration plus egg-fat” has a brain weight which is 6.4 per cent deficient. Thus the brain weight in the experimented group is — 6.4 less — 4.8 or 1.6 per cent lower than in the controls. This is the value entered in table 2. All the percentage differences in the accompanying tables have been obtained in a manner similar to that illustrated by the two examples just given. The only modification in procedure to which attention need be drawn is in the cases where the data from two series, 1913 and 1914, have been combined. In those cases the percentage deviation which is given in the table is the mean of the devia¬ tions for each series computed separately. GROWTH OF BODY AND ORGANS 5 GROWTH OF BODY IN WEIGHT The modifications of the growth of the body in weight due to the lipoid-free ration are shown in tables 1 and 2. Table 1 refers to the growth of the albino rats belonging to the 1913 series, while table 2 refers to the growth of the 1914 series. We note in both tables that the rats fed with the mixed ration made nearly normal growth in respect to their ages (see Donaldson ’06). The spring TABLE 1 Shoiving the weight of the body as modified by the lipoid-free ration compared with that of the rats raised on the mixed ration ( 1913 series) MALES FEMALES DATE Mixed ration (3) Lipoid-free ration (4) Mixed ration (4) Lipoid-free ration (5) 1913 April 16 . 94.7 93.8 85.0 76.2 30 . 129.7 122.7 112.0 92.4 May 7 . 137.7 127.8 125.7 98.6 14 . 153.7 134.8 146.7 108.4 21 . 149.3 136.5 litters 107.4 28 . 159.7 139.0 U U 108.4 June 4 . 166.3 140.7 u u 108.4 11 . 173.3 131.5 129.0 103.8 23 . 185.0 124.8 140.7 107.8 30 . 196.7 134.2 143.3 109.0 July 7 . 209.7 139.7 151.0 111.6 14 . 223.3 143.2 156.7 111.0 21 . 222.7 149.2 161.0 107.2 28 . 229.0 151.0 166.0 111.8 August 15 . 243.3 155.0 167.7 118.6 September 1 . 249.7 153.5 172.7 125.2 rats in 1913 made much better growth than the autumn rats in 1914. On the other hand, the experimented rats in both series made a noticeably poor growth when contrasted with the controls. In the 1913 series we notice that the experimented rats made continuous and steady growth throughout the period of experi¬ mentation, although the total amount of growth in weight was very slight. Curiously enough the experimented rats belonging to 1914 made a still smaller growth, and indeed in some cases the final body weight is no higher than the body weight at the begin- 6 SHINKISHI HATAI •e H -si £ e 5 g I ■ © •S' CO 'S >5 rO

ONXiC(NNOONM O N O iO O S 2 ■fS t-J d °»S *7 too, _ _ T3 MN ■S®w hJ c3 CM 00 ^ tF *0 ^ *0 0*0000100000010*0*0 10*0 o s e 0*00*0*0000*000*0*0*000 C50^CDCO^WC^CONiC^N(NH03 t^oooooooooooooooooooot^ co o co o CO O CO N o o co co O (M N O O ’ — 'O’—! CM a3 .O n a a « s > o o a £ p >> >> s-l c3 c2 42 3 fl .S fH Cj a QJ •rH *“5 P=H 2 GROWTH OF BODY AND ORGANS 7 ning of the experiment. This difference in growth in the two series may probably be due to the different physiological con¬ dition of the rats in these two series, combined with slight differences in the preparation of the ration. One point is clear, however: that the rats cannot continue the normal rate of growth on the lipoid-free ration in combination with the salt mixture which was used. In table 2 we have also the data on the growth of the body of the albino rats which were fed first with the lipoid-free ration and later with the same ration to which a minute quantity of the egg-fat had been added. For convenience, these last mentioned rats will be designated simply as ‘egg-fat series.’ It was found by McCollum and Davis (’13) that the rats whose body growth had ceased for a long period as the result of the lipoid-free ration, could be made to grow by the addition of a minute quantity of the extract of egg to the experimental ration. In order to see whether or not the rats thus treated would show any modifications other than those shown by the rats fed with the simple experimental ration, a small series was carried on. As will be seen from table 2, the 1914 rats given the extract of egg did not show the improvement in the growth of the body which was to be anticipated.1 Thus the growth of the body is nearly identical in both the lipoid-free series and in the egg-fat series. Why in the present experiment the egg-fat series did not show a noticeable improvement in the growth of the body is not clear. However, from the fact that the control rats belonging to the 1914 series did not make satisfactory growth when contrasted with the 1913 series, we conclude that the failure to grow was 1 “Our experience in feeding synthetic rations in this laboratory has con¬ vinced us that there exists a great variation in the vitality of individual rats as indicated by their ability to grow on such rations. It is unfortunate that practi¬ cally all of the animals employed in the work here reported were not sufficiently vigorous to grow for a time in a nearly normal manner on the experimental ration, or to respond by a period of active growth when the ration was supplemented with egg yolk fats. We have individual rats in our colony at the present time which have been on the diet employed in the lipoid-free period with egg yolk fats added, during more than six hundred days, and which compare favorably with our stock rats in size and well-being.” — E. V. McCollum. 8 SHINKISHI HATAI probably due to a peculiarity of the rats rather than a peculiarity of the experimental ration. Osborne and Mendel (’12) obtained normal growth of the rats with the ration from which the lipoid had been almost entire¬ ly removed. They carried the experiment for a considerable length of time by beginning with albino rats slightly over 30 days in age. In one series the experiment lasted for nearly 160 days. In every instance, so far as one can judge from the graphs, the body weight of the experimented rats was nearly identical with that of the control rats, while McCollum and Davis’ rats, fed with the lipoid-free ration, did not grow at any period to the size of the controls (McCollum and Davis T3; see also present series) . This difference in growth between the rats of Osborne and Mendel, on the one hand, and those of McCollum and Davis on the other, was undoubtedly due to the nature of the inorganic salts and some extracts still contained in the food. The Osborne and Mendel rats received the inorganic salts from protein-free milk, while those of McCollum and Davis received the salts which were a laboratory mixture of pure chemicals. In refer¬ ence to the varying effects of different salt mixtures McCollum and Davis state (’13) that “Young rats have been found to be very sensitive to variations in the character of the salt mixtures supplied, but with certain mixtures we have been able to obtain practically normal growth for periods varying from 70 to 120 days. Beyond that time little or no increase in body weight can be induced with such rations. The rats may remain in an apparently good nutritional condition on those rations for many weeks after growth ceases.” ANATOMICAL ANALYSIS We now wish to present the results of the anatomical exami¬ nation of these interesting rats reared by McCollum at the University of Wisconsin. Although the growth rate was dissimilar in the two consecutive years, nevertheless it was found that the alterations shown by the various characters are nearly identical in the two series of experiments, and on account of this uniformity in the results, as well as to avoid unnecessary complication by presenting the GROWTH OF BODY AND ORGANS 9 two series of data separately, I have combined the results. Con¬ sequently, unless otherwise stated, the figures given in the tables represent the averages of the two sets of data belonging to the 1913 and the 1914 series combined. CENTRAL NERVOUS SYSTEM If the lipoid-free ration is able to produce any alterations in the lipoid content of the organs, the central nervous system would naturally be expected to indicate such effects, since the central nervous system of the albino rat at about 200 days of age normally contains some 60 per cent of lipoid in the dried residue (Koch ’13). This lipoid content is certainly greater in the nervous system than in any other organ (Koch ’ll). The weights of the central nervous systems of the experimented and of the control rats are shown in table 3 (see also page 16). As will be seen from this table, the weight of the brain with respect to the body length is generally slightly smaller in both the lipoid- free and egg-fat series. Only one exception is found in the female rats (B) fed with the lipoid-free ration in which the experimented rats show a slight over weight of 0.7 per cent. This slight increase is probably due to the abnormally small brain weight of the control rats, thus raising the relative weight of the central nervous system of the experimented rats. Indeed the normal brain weight of the female rats corresponding to the body length of 189 mm. should be 1.80 grams as against the observed weight of 1.73 grams, i.e., the observed weight of the control is nearly 4 per cent less than the normal brain weight. Without making any correction, however, we find on the average that the experi¬ mented rats show about 2 per cent less brain weight than the controls. Similarly we find a reduction of 2.1 per cent in the weight of the spinal cord when compared with that of the control rats. This reduction of 2 per cent in weight in both the brain and spinal cord is somewhat greater than what we might expect from the normal fluctuation, nevertheless it is certainly far smaller than one might anticipate from the nature of the experiment. It seems reasonable, therefore, to conclude that the central nervous TABLE 3 Showing the various measurements of the rats fed with the lipoid-free ration compared with the control rats 10 SHINKISHI HATAI fc PS H W O H « j£ a > £ „• w £ % o o 04 CO 00 00 H 00 M 3 O O I I- lO o o CO CO ^ T}H CO 00 04 rH 05 CO ^ CO 04 rH c3 S-. 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O o O o o -V CO p* <55 <30 CO tO 04 00 05 CO 05 1 - 05 05 CD CD o o ■*— i o o O o o o 05 o o CO o o co o o tO <55 o o CD o o o <55 | OS 1 O 1— 1 e g <: 1 -t’ CO f^5 o T - 1 rh o CO 00 o CD CD "Hi 05 -t l- 1- CD CD o o CD g o o O o o o 04 Is o o CO o o CO O 1 1 £-» 1 fe; I- CD 05 05 1- 00 CO *o 00 to ^/“S 04 04 o o 05 o o GO1 o o tO o o to o o y—l o o 1 04 Oi 1— 05 00 Y— 1 1—1 CO 1- GO CO — r CD CO 04 CO OJ o o o o o 04 o o b o o GO o o O o o 04 1 e -r 05 05' G5 CD GO o 00 I- C5 s .1 T—> i-H i— ■< , .2 d C x X c3 X D £ w £ r-* D w cJ o ■_■ c O *J o .2 o 4-3 o o c ^ Q) a -4-3 «4-H c 4-3 *v o £ o o b£ b£ « o c g3 X r< Lipoid- Per c o S X 2 Lipoid- Per c O S o .2 c« Per c o o I TABLE 6 — Continued GROWTH OF BODY AND ORGANS 19 Cfi n w w 5? P Z H H a S *< 2 ce Eh a If 2 ci -h r fc Hioxa'i ACIOtl iO