g ': rm III. III III M I I III I I I III INHERITANCE OF BENT-NOSE IN THE RAT [RATTUS NORVEGICUSI WITH OBSERVATIONS ON THE EFFECT OF NUTRITION % TEE 2'7.) 8 FOR THE DEGREE BF II. S, WaIter E. Hcston 1934 I A ‘ I fihqgnvcld“ (g, Ann-u... 4 ._ $ §\ manor 0? BRIE-NOSE I]! THE RAT (RAHUS IORVEGICUa) II'I'H OBSERVATIONS 01' THE EFFECT OF NUTRITION Thu is Submitted to tho Faulty or the lichlgan Btute College in Partial Fulfillment of the Requirements for the Degree of later of So ions. by r Ialtor l. gglton Hay, 1934 1,5.7; b ACKNOILEDGWS The author wishes to express his appreciation and thanks to all who were of assistance in this work. 'Special gratitude is due Dr. H. R. Hunt who suggested the problem and under whose careful guidance the work was done; Ir. I. D. Burrington who gave many helpful suggestions concern- ing nutrition; Dr. 0. A. Hoppert of the Chemistry Department who gave information on the line teat, bone ash determination and rickets 3 Professor 0. L. Snow and Ir. Richard Struwin of the Physics Department for their aid in taking the x-ray pictures; and Professor I. E. Reuling of the Engineering Department for his aid in devising measuring instruments. H- , Q P.2- rm a: a II III TABLE OF CONTENTS Introauetion .0.........OOOOOOOOOOOOOOOOOO0.0.0.0.... History of the Bent-nosed Rat ....................... General Survey of the Study of the Bent-nosed Rat ... Part I -- Anatomical Study of the Bent-nosed Rat .... 1. 2. 3. 4. Purpose of the Anatomical Study ............... Study of the Skeleton other than the Skull .... a. The x-ray Study ......................... b. Study of Whole Skeleton ................. Study of the Skull ............................ Conclusions on Anatomical Study ............... Part II -- Observations on Effects of Nutrition ..... 1. 2. 3. 4. 5. 6. First Period - Results of the Varied Diet ..... Second Period - Results of Fox-chow Diet ...... Third Period - Results of Return to the Varied Diet ... Fourth Period - Results of Controlled Diet .... Tests for Rickets in the Bent-nosed Stock ..... a. The x-ray Test .......................... b. The Bone Ash.Percentage Determination ... c. The Line Test ........................... Conclusions Drawn from.0bservations on the Effects of Nutrition. QmmmO’II-NH GGUGH IFGNOH 36 39 42 43 43 46 48 Ilullillll!‘ lull III) I III . [I‘ll]. rrhl_'l|.llelllll..ty >,.»§l he} . I.... I’.‘ I VI VII VIII TABEE OF CONTENTS Continued Part III --Heredity of the Bent-nosed Rat ........... 1. Technique Used in Conducting Breeding Experiments .... 2. Presentation and Discussion of Data ........... a. Data Obtained During First Period ....... b. Data Obtained During Second Period ..... 'c. Data Obtained During Third Period ....... d. Data Obtained During Fourth Period ...... 3. Summary of Heredity Study ..................... General Discussion .................................. General Conclusions ................................. Bibliography ........................................ mlm‘tion or Plato, 0.0.0.0..........OOOOOOOOOOOOOO Page 50 51 52 33 55 55 60 63 64 66 67 70 IILDIYIIIIIIllqll' Table Table Table Table Table Table Table Table Table Table Table Table Table II III VI VII VIII II III XIII TABLES Summary of Mr. L. W.‘Iiren's Breeding Experiments ................................... Skull Measurements and Indices of Bent-nosed Animals ....................................... Skull Measurements and Indices of Normal humus..u.u.u.u.u.n.n.u.u.u.u.u.. Comparisons of the leans of the Five Measure- ments lads of the 66 Bent-nosed Animals and the 78 Normal Animals ......................... Comparison of Indices of Bent-nosed and Normal Skulls ................................. Comparison of Skulls Bent to the Left and Skulls Bent to the Right ...................... Indicating Age at which Animals could be Transferred from Fox-chow to Varied Diet and Develop Bent-nose ......................... Results of Bone Ash Percentage Determination .. Animals Born Before January 1, 1933 ........... Animals Reared on Fox-chew .................... Animals Born after lay 5, 1955, and Reared on Varied Diet ................................... Percentage of Bent-nosed Progeny with Decreasing Proportion of Bent Ancestry ........ Animals Born after November 18, 1953, and Fed on Controlled Diet ........................ Page 15 2O 26 27 30 37 45 54 56 57 59 62 Figure 1. Figure 2. Pigure 3. ILLUSTRATIONS lalfonmed Scapula of Bent-nosed P2 6‘444 ....... Points A, B, O, D. B, F, G. H) I, and J, on which.8kull Measurements were Made .......... Negatives of the Line Test with one Standard Rachitic Negative for Comparison ............... 13 49 Plate I Plate II Plate III Plate IV Plate V Plate VI Plate VII Plats VIII Plate 11 Plate x Plate XI Plate XII Plate XIII PLATES External appearance of a bent-nosed animal .... Skulls showing the bent-nose condition ........ Instruments for measuring skulls .............. Demonstrating the method used in measuring the angle of bend in skulls ................... x.ray phOtograph or normal 6‘ 1001 0 e e e e e e e e e e e 0 X-ray photograph X-ray'photograph X-ray photograph X-ray photograph x-ray photograph X-ray photograph Xpray photograph Xeray photograph of bent of bent of bent of bent of bent of bent of bent of bent dlBOOOOOOOOOOOOOOOOO P2 9 351 eeeeeeeeeeeee. tee 575 12 (7369 6'53 ................. 9 103 6‘63 ................. €193 Page 71 72 73 74 75 76 77 -78 79 80 81 82 83 ..PII' In... Pu III I INTRODUCTIOI Our most important problems often are encountered during the study of anomalies which at first appear cpite simple. He can never predict with accuracy the nature of these problems nor the field in which they will occur. The bent-nose condition of the rat was first attacked as a simple lendelian trait, but it has since appeared to involve more cmplicated genetics as well as factors which are not of a genetic nature. ~ The name “bent-nose“ was chosen because it describes the actual condition encountered in the rat. As the me im- plies, the nose is bent to one side. This bending may be to the right or left and to a varying degree. No other abnormal- ities occur. Plate I shows an adult bent-nosed animal, while Plate II presents skulls of such individuals. It is interesting to note that the character does not appear at birth. Throughout the experiment, the youngest animal to show the character was thirty-four days old. others have not manifested it until they were 120 days old,and still others were not found to be bent until their skulls were examined after they were full grown and etherised. The author has found no other case of this character, nor any similar character, described in the literature. other skull abnormalities such as hydrocephalus and “high-brow" of the rat have been studied, but none are comparable to the bent-nose condition. However, a few cases of a similar character have been observed by Dr. R. I. Peldmsn and Ir. Alan L. litchell of the University of Michigan among their mice which have an ear fibnomalitYe Th... Obflflflntimfl 111?. not 15¢an nah14-1-nul HISTORY OF THE BENT-HOSBD RAT The ”bent-nose" character of the rat was first dis- covered in the elementary Zoology laboratory here at lichigan State College in 1930. Rate from the rodent colony here are used for study of the malian skeleton. During the spring of 1930 several of the skulls which were being studied were found to be bent, the bend being so evident that it could not escape notice. Immediately an investigation was begun by Dr. H. R. Hunt to see whether the condition could be found in any other animals of the colony. In a red-eyed-yellow strain derived from Dr. E. E. Castle-s stock, the abnomality was quite preva- lent, but it did not occur in any of the other stocks of the colony. The author since found it occurring in a meager strain of black-hooded agouties which were believed to be related to the red-eyed-yellows. However, it was never found in the other colony strains: the-black-hoodeds and the pink-eyed-yellows de- rived from Dr. I. B. Castle's stock, nor the blacks descended from Dr. C. A. Hoppert’s animals. These three strains were not known to have been related to the red-eyed-yellows. During 1931 and 1932 Ir. L. I. liren attempted to de- termine whether or not the character was inherited. He made nine P1 matings of bent x normal animals from the bent stock and two matings of- bent x bent. The data of Ir. liren's work are given in Table I. Table I SUMMARY 01" IR. L. I. IIREN'S BREEDING EXPERIMENTS Type of Hating No. of Total lomal Bent mat ing; progeny progeny progeny P1 lat ing Bent x Nomal from 9 63 54 9 bent stock Bent x Bent 2 '7 '7 0 As can be seen in the table, nine bent-nosed: animals 'in a total of 63 appeared in the ’1 generation. This would tend to indicate that the character was inherited, possibly as the result of interaction of several genes. lots that the seven progeny from the bent x bent matings were all normal. This concluded hr. Iiren's work. At this point the author first attacked the problem. Gm SURVEY OF THE STUDY OF THE BENT-NOSE!) RAT The fact that the character was so prevalent in one strain but occurred in none of the other strains of the colony indicated that bent-nose was inherited. The author initiated an experiment to determine definitely whether the character was or was not inherited, and to study its mode of inheritance. During the fall of 1932 P1 matings and matings of Iiren's animals were made. Backcross and P1 matings were plan- ned to follow according to the conventional method of analysing a genetic character. However, the train of progress has since been derailed so often that no definite plan could be consist- ently followed during the analysis of the problem. The first complication encountered was the result of a change in food on January 1, 1933. Before that time the animals had been fed on a varied diet, getting cooked corn meal one day, possibly cooked wheat the next, and perhaps rice pudding the third, depending on what the man who prepared the food do- cided to give them. On January 1, 1933, fox-chow was introduced as a food. This is a well balanced diet manufactured in a ' pellet form which can be very conveniently fed, convenience in feeding having been the primary reason for adopting it. The new diet brought about a very important change in the frequency of bent-nose. Until January 1, 1933, five animals out of a total of 112 from the various matings were bent. Io animals reared on fox-chow developed the bent-nose, though in all. 191 animals were reared with this food. ....“ U.‘ Fox-chow was continued until lay 6, 1933. At this time, the author returned the breeding stock to the old ration fed previous to the fox-chow. Other matings favorable for pro- ducing the character were also made. The results of the change back to the old diet were amazing. Bent-nose animals began to appear. in canparative large numbers. In the 27 progeny of four inbred crosses between normal animals from bent-nose x bent-nose matings, 67% were bent-nosed. However, these results among animals fed on this un- controlled diet could not be used as definite proof that the character was hereditary in nature. The food was so coarse and so incompletely mixed that there was a chance for individuals to eat only certain constituents. It was possible that those rats which were developing bent -nose were selecting a deficient diet. To eliminate this possibility a controlled diet was made by finely grinding and thoroughly mixing all the different ingredients that had been fed during the period in which the character appeared. The stock was transferred to this controlled diet on November 18, 1933, and there it has remained until the present time (lay, 1934). Three phases have appeared in this problem. Nutrition appears to be quite as important a factor as heredity. The third phase of the investigation is the anatomy of the anomaly. The body of this thesis will deal with each of these phases separately. First, the anatomy of the bent-nosed rat will be taken up. Second, nutrition in relation to bent-nose will be discussed; ami third, the author will deal with the inheritance of the trait. PART I ANATOIIGAL STUDY OF THE BERT-HOSE RAT External observations of 'bent-nosed“ animals showed that they were very much like their'normal sibs except that their noses appeared bent to the right or left. The body in general appeared quite normally healthy, the only exception being that the animals often were a little underweight. This could be attributed to their slight handicap in eating. The lower Jaw was nominal, and, since the nose was diverted, the incisor. teeth did not articulate as they would normally. The point at which the nose bent appeared rather definite externally, being a little anterior to the eyes, but the degree of bend was quite variable. In some animals one had difficulty in detecting any bend while others showed a very noticeable deflection. In general, the heads of the bent animals appeared shorter. An extremely afflicted animal gave the impression of being very “pug-nosed". Such a condition of shert-headedness has been found in a few of the normal animals. A more thorough analysis of this condition will be discussed later. Purpose of the Anatomical Study It was evident that external observations told us little about the csseous anatomy of the 'bent-nosed' rat; so a thorougi study of the skeleton was made. -7- Often one bone abnormality is accompanied by others. It is quite reasonable to suspect that any set of factors caus- ing one bone anomaly would cause others in different parts of the body. Rickets may make a child bow-legged, and this be accompanied by a curved spine or sternum. Abnormal vertebrae in the lumbar region have been found in flexed-tailed mice. Thus it was important to determine whether or not other bone abnormalities accompanied the bent-nose condition in the rat. A careful study was made to determine Just which bones of the skull were bent. Ias it the nasal bones or the frontals that were deflected? Did this affect the maxillaries or the pre-maxillaries, and were there any bones posterior to these which were included in the bending? As before mentioned, the bent-nosed animals' heads appeared musually short. It was desirable to make a more careful study to determine if the skull was abnormal in res- pects other than having a bent-nose. Ias the skull relatively short, wide, and deep? Did the bending of the rostrum deform other bones? In other words, in how many respects did the skulls from the bent-nosed animals differ from normals? It was observed that the skulls were bent either to the left or the right. A study was made to determine the fre- quency with which they were turned to the right. Related to direction of bend was angle of bond. Ihat were (:13. range .111 mean degree of flexure? Also, were those which were deflected to the right bent to a greater degree than skulls bent to the left? These were the questions whose answers were sought in the following anatasical study. -8- Study of the Skeleton Other Than the Skull. This study of the skeleton as a whole was made by two different methods: (1) by x-ray pictures, and (2) by cleaned whole skeletons. The structure of bones can be studied quite well in a good x-ray picture. However, it is desirable to supplement this with a study of the actual skeleton. The x-ray study. X-ray photographs were made of eight bent animals and, for comparison, of two normals. These photographs are included in plates V to XIII inclusive, with the exception of one of the normal animals. The technique in taking the x-ray pictures was as follows: First the animal was anesthetized with ether. Care was taken not to give enough to cause death. The rat was then tied to a board, back down, with the four legs stretched out as shown in the pictures. A cloth muzzle suspended on two wire loops was placed over the nose to hold the head down in place. The film was slid in between the animal and board and both were placed under the x-ray tube at a distance of 30 inches. For four and one-half seconds the rat was subjected to the rays of the machine which was set at 4.6 If (filament current), 20 LA. (milliamperes, and 58 LY. (kilo-volts). The films were then taken to the dark room where they were opened and immersed in the developing fluid for five minutes and then in the fixing fluid for five minutes or longer. A careful detail study was made of these photographs, comparing the bent-nosed with the normal animals. F2 8‘ 375, -9- F2 3369, 6' 13, 61%, and 2 103 appeared to have curved spines, the condition being most pronounced in 9 1013. r2 d‘ 575, Pa 6‘ 369, and 6' 13, which are still in the breeding cages, have been examined, and by palpating their spines one can detect no curva- ture. 9 103 am! 6' 193 were etherized and on the examination of their spines no abnormality was found. The appearance of curved spines may be explained as follows. than the anismls were tied back down to the board their muscles had a tendency to comract. As the four legs were stretched out tight the spine was pulled out of shape. The fact that the head of 9 108 is thrust caudad against her body tends to verify this reasoning. All the other bones of the bent animals appear to be nomal with the exception of the rostrum, the deflection of which can be easily detected. Study of whole skeleton. The skeletons of eight bent-nosed rats were cleaned and thorougaly studied. The animals were boiled for about 20 minutes in a soap solution which loosened the flesh, making the cleaning of the bones quite easy. tith one exception, no bone abnormalities aside from the bent skull were found in these aninls. An abnormal scapula was observed in one case. The scapular blade was bent radially causing the acromion process to be elongated. Il'he scapular spine was much mcm-e prominent than normal. The opposing scapula was normal. Figure 1 shows an anterior and a lateral view of this abnomal bone. Since only one anomalous scapula was found in the 16 bent-nosed animals examined as whole skeletons and by X-ray photographs, one could not conclude trmt this peculiarity was related to the bent-nose character. One migit find one such aberrant bone in an equal number of normal animals. Lateral view (g x 3 Posterior view ‘Fig. l Halformed scapula of bent-nose F2 6'444. -11- From these two studies one must conclude that the fac- tors causing the bent-nose condition are not general, but quite specific in their expression. They cause the anterior bones of the skull to become bent, but do not alter other bones in the (body. This would indicate that the character was influenced by heredity as one would expect the anomaly to be accompanied by several other defects were it caused by a dietary insufficiency acting alone. This point will be discussed further in Part III which deals with heredity. M“ the Skull Sixty-six thoroughly cleaned bent-nosed skulls and ‘78 normal ones from the experiment were the subjects of anatomical study. These were selected at random from P1, F1, and F2 animals, and the progeny of backcrosses. First the bent sknlls were studied to determine which bones were affected. It was found that the angle of bend was . quite definite, occurring at or Just in front of the suture be- tween the nasal and frontal bones. The bones affected on the dorsal side were the nasal, premaxillaries and maxillaries. The aygematic arches appeared normal. (See plate II.) On the vent- ral side of the skull the bend seemed more uniformly distributed from me basisphenoid bone to the incisors. The bones in the posterior region, the occipitals, parietals, inter-parietals, squamosals, the tympanic bullae and the petrosal bones all ap- - peered normal. The anatomical study included also definite measure- ments on all 66 bent-nosed skulls and 78 normal ones. The .12- measurements of the two types of skulls were compared. (Tables II and III.) Five major precautions were taken for accuracy: (1) All skulls were thoroughly cleaned of flesh in order that the points for measurements would be well defined. (2) All skulls were complete. (3) The skulls of no animals less than 100 days of age were used. (4) The skulls were dried for at least two weeks in a mean temperature of approximately 130° F. (5) Points for measurement were selected where the bone was relatively thick. The following measurements were made. (Fig. 2.) The length AB was defined as the distance from the dorsal border of the foramen magnum to the most anterior point of the upper in- cisors. The width CD was taken from a point on the squamosal bone Just dorsal to the sygomatic process of the squamosal and directly ventral to the descending portion of the suture between the squamosal and parietal bones, to the sen point on the oppos- its side of the. cranium. A posterior thickness HF was taken as the distance from the point P. at which the sutures separating the two parietal bones and the interparietal bone unite to a point P midway between the two tympanic bullae on the suture between the basioccipital and the basisphenoid bones. An anterior thickness OH was measured. This measurement was defined as the distance from the point G at the union of the sutures between the frontal and nasal bones to the point H which is even with the anterior border of the first molars on the suture between the maxillaries. The fifth measurement was the width outside the -13.. Dorsal view Ventral view Lateral view Fig. 2 'Points A, B, c, D, B, F, G, H, I, and J on which measurements were nude. Length AB Posterior thickness BF Iidth CD Anterior mickness OH lidth outside 1st molars IJ -14- first molars, or IJ. I and J were the most distant opposing points on the lateral borders of the first molars. These distances were accurately measured to .01 inch. The apex of the angle of bend was first marked with the fine line of a 5H lead pencil across the internasal suture. The skull was then placed on a surface plate between two steel blocks with.square faces, making sure that points Aland B articu- lated squarely with the faces of the steel blocks. One block was only slightly thicker than the skulls. This minimized the error of parallax. The length was calculated as the sum of the dis- tances from.the faces of the steel blocks to the apex of the angle. A magnifying glass was used to make this reading accurate. The other measurements were made with a strain gage so set up that the skull could be placed between two steel points, the dimension being measurable to .001 inche This apparatus is shown in plate III. The date thus gathered.were attacked with.the hdea of determining differences and likenesses between the normal and the‘bent-nosed skulls. It is interesting to compare the means of these five measurements. (Table IV.) 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ANIMALS AND THE '78 NORMAL ANIMALS lleaenronent Bent-nosed Normal Difference _e.ninnle animal; — Length AB 1.49? in. 1.561 in. .064 in. lidth CD .576 in. .584 in. .008 in. Posterior thickness RF .415 in. .420 in. .005 in. Anterior thickneu GR .596 in. .598 in. .002 in. 'Mtgo‘l’fizigg 1” .344 in. .539 in. .005 in. -27- 00H H mm 93.0 mmmdama we; aim 04 3.03.2. mm; ,_,2..m mm; 3.03.? n35 £2; rumongvans Hofinopmom Illnblll. . . 00H H mm mm o H¢.o Hw o odm N5" m.m ¢¢.mflm.mm mm.m rm.” H.& 3.0 «p.90 HounH ~3sz nmmonxowna nofinopgm . .. m4 #H p dH.onfio.H $0.0 mN.H m.o mmo.onflm.bn om.o >N.H m.o mOH.onfim.mn 00H fi no “3an 533-53; noapop noapon .223 not... .233 .33.. on» Ho \0 $59 832 of. Ho \o :33 :32 .n.m momma .mAA mums .a.m macs .HHQ noospon HocmH afipam nonunomofin mp dupe» om Hope» uHHdMn Huanoz mHHde comonnpqom mg 93582 a: gmozlazfim .mo mmfioHQZH mo zomHM4mEoo b 35mm. A more reliable comparison is by means of indices, which are the ratio of one measurement to another. This is the method used in the study of AnthrOpdmetry. Alei Hrdlidka, in his work on.Anthr0pometry (1920), defines several different indices of the head and skull. The cephalic index is Br 12% 111 100. By this index humans are classified; one having a cephalic index up to 74.9 is classed as dolichecephalic; 75.0 to 79.9, nesocephalic, and 80.0 and above, brachycephslie. Other indices for the skull are: lean Height Index - m : Height-Length Index - W . Not only in the study of skulls are indices used but also for other bones of the body, as the femur, the scapula, or the sternum. Three indices were calculated for comparing bent- nosed skulls with normals, namely: (1) lidth-Length Index 92.15292 (2) Posterior Thickness-Width Index W (5) Anterior Thickness-Iidth Index 21%}22 These different indices are tabulated in tables II and III, while the results of the comparative study are given in table 7. Note that the one index only, the Iidth-Length.index, shows a statistically significant difference between the bent- nosed and normal skulls. The results prove that. the bent-nosed skulls are shorter in relation to the width than are the normal skulls. This is not due to the variation in the width.measure- ment, since the means of the other two indicea do not show a statistically significant variation. Since the method used in measuring the bent-nosed skulls was not the same as for the normals, one questions whether or not this variation in length was due to faulty measuring of the bent-nosed skulls. It is hardly probable that this is the case. The mean Width-Length Index for the normal skulls was 57.5. The mean width of the bent-nosed skulls was .576 inch. Therefore, a mean length of 1.556 would be necessary for the bent-nosed skulls to give a Width-Length Index identical to that of the normal skulls. This would be .059 inch greater than the actual mean length. It is highly im- probable that an average error of .059 inch was made in measur- ing the bent-nosed skulls. Iith successive measurements of the same skull the error never exceeded .01 inch. The next phase of this skull study was centered on the bend of the skull, the direction, degree and variation of the deflection. Table VI gives the data for this study. A method was devised for measuring the angle of bend by means of a glass straightedge and a protractor. (See plate IV.) The skull was placed on a mass of plastic clay and pressed down so that the clay held it firmly. The aagittal and frontal sutures were then placed in line with a glass straightedge which was held firmly by a clamp on a ring stand. lith the apex of the angle, taken at the suture between the nasal and frontal bones, the protractor was brought in line with the distal end of the internasal suture. The ngle of deflection was read to the nearest degree at the point where the pro- tractor crossed the straightedp. Analysis of these data reveals the following points. (1) The degree of flexure ranges from as low as 4° to 55°. It is interesting to note that these two extremes were 15 animals. (2) The mean degree of flexure was l7.5°. (5) The frequency of the left bends was not signifi- cantly different from the right. or the 66 animals, 51 bent to the left, and 55 to the right. (4) lo statistically sigificant difference occurs between the mean angles to the left and rigzt. This comparison is given in table 71. rants v1 00104111303 or slums BENT T0 m LEFT 11m SKULLS BENT TO THE RIGHT lean angle lean P. B. Dif. of Ratio of bend Devi- o’ of the loans Dif ation Distri-_ 3,—3- bution '3‘ Skulls bent ' to 1m: 17.3% «13° 4.71° s.1s° 4.1s° Total 51 0.5% 1.016 0.49 Skulls beat 0 e c to right 17.: a: 4.1s° 4.56° 5.11 4.12 Total 55 Conclusions on Anatomical Study. (1) The expression of the factors causing bent-nose in the rat is centered on the bones of the anterior region of the skull. lo other bone abnormalities have been found normally accompanying this bent-nosed condition. -31- (2) The mean Width-Length Index is significantly greater for the bent-nosed than for the normal skulls. That is, the length is less in comparison to the width in the bent- nosed than in the normals. (5) lo simificant difference occurs between the mean Posterior thickness-Iidth Indices, and the same is true of the , mean Anterior thickness-Width Indices. (4) The range of the degree of flexure is from 4° to 35° with a men of 17.5". (5) lo sigificant difference is found between the number, or mean angle of those animals bent to the right and those bent to the left. -52- PART II OBSEVATIONB 0N EFFECTS OF HUTRITI ON The boundaries of the science of Genetics will never be truly defined, any more nearly than will the boundaries of lutrition, Physiology, Chemistry or any other science. Genes cause the expression of characters only with the cooperation of all necessary physiological and environmental factors. Thus, we have the different sciences overlapping. when one attempts a genetic problem he is likely to find.himse1f automatically carried into the adjoining territory of any of the other fields, and Just where one crosses the boundary cannot be determined. lhen the author began the study of the bent-nosed rat, he had no intentions of making a nutritional study, but he had not progressed far until some marked effects of the change of diet became evident. 0n fox-chow no bent-nosed animals appear- ed, while on a.mixed diet a certain percentage of animals ap- peared bent, this percentage varying somewhat with the type of matings of which.they were progeny. The author regrets that a thorough analysis of the effects of nutrition on the bent-nose character has not been made. Such an analysis will be necessary before all the genet- ics of the problem.can.be understood. However, some of the observations on the effects of different foods merit further discussion; also some tests for the determination of rickets will be discussed. The experiment could be divided into four different periods according to diet: (1) From the beginning of the work in September, 1952 to January 1, 1955, during which time the animals were fed on a varied diet. (2) The period from January 1, 1955, to lay 5, 1955, when fox-chow only was fed. (5) 0n lay 5, 1955, the animals were returned to the imperfectly mixed varied diet again. This period ran to lov- ember 18, 1955. (4) From lovember l8, 1955‘to the date of writing (lay, 1954) the animals were fed on a controlled diet. Bach period will be discussed separately, dealing with the types of food and the results of feeding each" type.. First Period - Results of the Varied Diet. Then the experiment was begun, the animals were kept on the cooked diet that they had been fed for the past several years. This diet consisted of a great variety of grains in- cluding corn, oats, wheat, barley, and rice, all these in their many different preparations. It also included some vegetables such as potatoes and carrots with lettuce and cabbage occasion- ally. Raisins were added for fruit, and now and then cocoanut was mixed in. Some meats were fed, this usually being salmon, though occasionally meat scraps were added. Cod-liver oil was furnished about once a week, about a half cupful being poured into the 8 or 10 pounds of feed for one day. The animals could detect this, and the feed with cod-liver oil in it was seldom eaten as well as that without. t-‘ith all these different foods, the rats could have had a good diet had not they have been fed cooked corn or cooked wheat too often due to the ease with which they could be prepared. During this period 112 animals were born, five or 4.5% of which developed bent-nose. (Table I!) These 112 were frm various types matings such as F1 (progeny of bent x nor- mal mated inter se), backcross (F1 x bent), and P1 (bent x normal), all of which will be discussed later. More rats migit have developed the bent-nose condition had not they have been put on the fox-chow food while still quite young. As the reader will recall, the bent-nose character does not develop until the animal is about 52 days old or older. Second Period - Results of Fox-chow Diet. As previously mentioned, the stock of the colony was put on the fox-chow diet for convenience. Small wire mesh con- tainers were made which would hold a week's supply of food, thus eliminating the daily chore of feeding. All experimental animals were put on this diet January 1, 1955. Pox-chow is a quite well balanced diet in pellet form manufactured by the Purina Company in St. Louis, Missouri. It was evidently primrily intended for a fox food, hence the nama fox-chow. However it is relished by most rodents as well. The rats ate it readily. They grew and bred well on it. According to the manufacturers, fox-chow is composed of the following materials: meat (beef), wheat germ meal, dried skim milk, cod-liver oil, barley malt, blackstrap molasses, -35- cat cereal, corn, wheat bran, and iodised salt. These materials were mixed in proportions which gave the following percentages: protein 20% e fat 5% fiber 6% nitrogen-free extract 50% carbohydrates 56% Ash 6% The caloric value was calculated as 1500 calories per pound; the nutritive ratio was 1 to 5, and the calcium phosphorus ratio,'1.4 to 1. This would indicate a slight excess of phos- phorus in relation to the amount of calcium. The ratio of calcium to phosphorus in the body is usually considered to be about 2 to 1, and, therefore, the ratio of calcium to phos- phorus in a food should be about the same. A total of 191 F1 (progeny of bent x normal) and backcross (1'1 x bent) animals were reared to maturity (at least 120 days) on fox-chow. m 2_f_ t_h_e_g_e_ animals developed 3.92. bent-nosed character, as their skulls were all normal when they were etherised at 120 days of age or older. Eighty-nine P1 animals and 52 progeny of bent x bent or a total of 121 were reared on fox-chow to partial maturity ranging from 8 to 105 days of age. llone of these animals appeared bent while on the fox-chow ration. They were later put back on the varied diet, and some of the younger animals afterwards developed the bent-nosed character. This point will be discussed more fully in the next section. 4 Some fallacy occurs in these percentages as they total 141%. Third Period - Results of Return to the Varied Diet. On or about Ray 5, 1955, all breeding animals, F1 animals and progeny of bent x bent, were returned to the varied diet fed previous to the introduction of fox-chow. This was done in an attempt to regain bent-nose. In addi- tion to the crosses already started, four inbred matings were made with progeny of bent x bent matings which had been reared on fox-chow and were normal, and 12 P1 matings were made of bent-nose x normal animals from the bent-nose stock. or the 89 ’1 animals which were returned to the varied diet before reachirg maturity, four developed the bent- nose, while of the 52 progeny of bent x bent which were treat- ed in a like manner, six later became bent-nose. Iable VII lists these litters with the age at which they were returned to the varied diet. Note tint no animal which had been fed on fox-chow for mme than 52 days after birth developed a bent-nose. One animal in a litter of seven developed a bent- nose after having been fed fox-chow until it was 52 days old; two animals out of a litter of six developed bent-nose after being returned to the varied diet at the age of 15 days; two bent-nosed animals appeared in a litter of five after being returned to the varied diet at the age of eleven days 3 one animal in a litter of two deveIOped a bent-nose after being returned to the varied diet at ten days of age; and four out of a litter of five developed bent-nose after being returned to the varied diet at 8 days of age. This means that animals which were transferred to the varied diet while still nursing -37.. Table VII IIDICATING AGE AT WHICH ANIMALS COULD BE TRANSFERRED FRO! FOX-CHOW TO VARIED DIET AND DEVELOP BENT-HOSE Type of mating normal Bent-nosed Age in days at which offspring offspring offspring were trans- ferred from fox-chow __d_f‘_ _§_ ___<_:"__ __¥_ to varied diet. ’1 mating e 2 105 ’1 mating 5 l 95 Bent-nose x bent-nose 2 l 95 ,1 mating 5 91 P1 mating 2 2 89 ,1 mating 4 2 82 P1 mating l 5 79 ,1 mating l 2 79 P1 mating l 4 60 Bent-nose x bent-nose 5 55 P1 mating 4 4 49 P1 mating 4 5 42 Bent-nose x bent-nose 2 4 40 Bent-nose x bent-nose 4 40 -33.. Table VII Continued Type of mating loml Bent-nosed Age in days at which offspring offspring offspring were trans- ferred from fox-chow o" 9 d' 9 to varied diet. P1 mating 5 5 40 P1 mating . 4 4 56 P1 mating 2 4 l 52 Bent-nose x bent-nose 1 4 51 P1 mating 5 2 50 P1 mating 2 l7 Bent-nose x bent-nose 2 2 2 15 ’1 mating l 2 1 1 11 P1 mating l l 10 P1 mating l 1 10 Bent-nose x bent-nose 1 1 5 8 Total .. ... 111 10 * P1 matings were bent-nose x normals unrelated to the bent stock. llo animals appeared bent-nose while being fed fox-chow. were for the most part the only ones developing bent-nose. Forty-five percent of the animals transferred before 17 days of age developed bent-nose. A total of 204 animals were born after lay 5, 1955, and fed on the varied diet. One hundred twelve of these ani- mals were normal and 95, or 45%, developed bent-nose. pp: hundred seventeen animals, g£_which.§§, pp 44!, developed bent- Pourth Period - Results of Controlled Diet. On November 18, 1955, a controlled diet was introduced to enable the author to investigate further the inheritance of the character. (See part III.) Though the results of this diet were not so striking as of the other two diets they should be discussed. This controlled diet was formulated with two views in mind: (1) it was to contain the same materials as the varied diet of the third period but to be of known proportions; (2) the controlled diet was to be so finely ground and thorough- ly'mixed that there would be no chance for selection of food by the individual rat. The following materials constituted this diet in the proportions indicated: -49- laterial Propgrtion_by_weight Rolled oats ' 10 parts lhole wheat 10 parts Bran 10 parts liddlings 10 parts Corn meal 10 parts Barley 10 parts Rice 10 parts lacaroni 10 parts navy beans 5 parts Lima beans 5 parts Coccanut (shredded) 5 parts Raisins 5 parts Potatoes (pared) 10 parts Carrots (pared) 10 parts Salmon 10 parts lhole milk , 10 parts laCl 0.5 parts Cod-liver oil 1 tablespoonful nor each 14 pounds of food. The grains, macaroni, beans, cocoanut and raisins were thoroughly mixed and finely ground together. Each day ten pounds of this mixture was cooked into a mush with tap water and later the proper proportions of milk, potatoes, carrots, salmon and cod-liver 011m added. The cooked ' potatoes and carrots were run through a fruit press as was the salmon to make it possible to mix them thoroughly. -41- This diet contained all the materials fed in the varied diet of the third period. However, the prOporti one were not the same since those of the varied diet were not known. The amount of wheat and corn was probably greater in the varied diet than in this one. It is evident that this diet, though intended to be deficient, was too well balanced to produce many bent-nosed animals. By January 27, 1954, no bent-noses had appeared in 145 animals. At this time milk and cod-liver oil were elimin- ated from the diet. On February 9, 1954, one backcross animal was foundto be bent-nosed, and February 17, 1954, one progeny of bent-nose x bent-nose was observed to be bent. Also, an- other such progeny was found to be bent February 19, 1954. These- three animals, 2.11, from a total of 145 were all that developed the bent-nose while the controlled diet was fed. The 145 animals included Only these which had been reared to maturity (120 days of age), ethsrized, and had their skulls examined. During this fourth period four control matings of normal rats imported from the Wistar Institute produced 64 animals all of which appeared nomal externally. However, only 19 of these animals were over 120 days of age though all were 65 days old or older when the final observation was made (lay 12, 1954). -42- Tests for Rickets ingthe Bentenosed Stock. Sherman defines rickets as a condition in which the mineral metabolism.is disturbed in such a way that calcifica- tion of the bones does not take place normally. Three types of rickets may occur: (1) Low-phosphorus rickets - when there is an excess of calcium in proportion to the amount of phosphorus; (2) Low-calcium.rickets - an excess of phosphorus in proportion to the amount of calcium; and (5) Low-calcium low-phosphorus rickets - when.both.are reduced. Rickets is usually accompanied by one or more of several bone abnormalities, as crooked sternum (pigeon breast), knobs forming on the ribs at their union with the costal car- tilage ihich produces the rachitic rosary, curvatures of the spine, and bending of the longer bones causing bowed-legs or knock-knees in children. Thus, when one finds a bone abnormal- ity influenced by food, he immediately suspects rickets; as did the author when he discovered that the appearance of bent-nose was influenced by diet. The next logical step was to make some tests to de- termine whether these bent-nosed animals were rachitic. Quot- ing Sure (1955); 'At least four methods are used for detection of rickets experimentally: (l) x-rays; (2) total mineral and oalcium.content of the bones; (5) phosphorus content of blood; (4) line test.‘ Brief tests were made by three of these methods, each.of which will be taken up. There was no determdnation for phosphorus content of the blood. -43- The X-ray test. Eight animels were subjected to the X-ray test, the X-ray photographs of which are given in plates VI to XIII in- clusive. Plate V gives the photograph of a normal animal for comparison. These are the x-ray photographs which were used for the anatomical study, and the technique for taking them was given during the discussion of that phase of the problem. In examining x-ray photographs for signs of rickets there are several points for which one should look. The skeleton of a rachitic rat in general looks “scrubby'; the 'pigeon breast" character appears in the sternum; the ribs show the typical “rachitic rosary”; the spine is apt to be curved; and the long bones may be poorly formed with.en1arged ends. From.the plates one observes that none of these charact- eristics appeared in these pictures, with two possible except- ions. Three of the skeletons appear to have curved spines, but,as the author explained in Part I,he believes this was due to muscle contraction under anaesthesia rather than abnormal- ities in the vertebrae. The skeletons of the bent-nosed animals are not as large as the normal skeleton, but this dif- ference is not great encugh.to justify one's concluding that they were rachitic. Thus, the x-ray test indicates that at the time the x-ray pictures were taken the animals were not rachitic. The bone ashppercentage determination. Determination of ash percentage compared to percentage of organic matter has been used extensively as a test for rick- -44.. ets. It has been found that after fat has been extracted and the bones dried, normal bones will range from an extreme low of about 45% ash to 60% or above, varying with food, age and sex. Proper proportions of calcium and phosphorus in the food with ample vitamin D tend to increase the ash percentage. Also the ash percentage increases with age, and females tend to have a higher percentage of ash than do males. The ash percentage of rachitic animals is exceedingly low, often being below 30%. To mke this test, a normal and his bent-nosed sib 91 days of age, two normals and their three bent-nosed sibs 163 days of age, and one lactating bent-nosed 9 299 days of age were used. In accordance with the usual custom only the femurs were tested. The animals were etherized and their fenmrs removed. After all the flesh had been rubbed and scraped from the bones the fat was extracted with alcohol for 50 hours, that being more than ample time. They were then dried and weighed. For three hours they were burned in a com- bustion furnace after which they were weighed again. From the weights before and after igniting the ash percentage. is cal- culated. The results of these procedures and calculations are given in Table VIII. Precautions for accuracy, as handling crucibles with tongs rather than fingers, using crucibles at constant weight, etc., were observed throughout the analysis. Bent 8' 1501 and normal 6" 1506 had been fed wrough- out on the cont rolled diet. Isle 1501 had. appeared bent Feb- ruary 1’7, 19:54, and 24 days later was used for the ash percent- ag test. No significant difference appears between the ash -45- Table VIII RESULTS OF BONE ASE PERCENTAGE DETERMINATION Animal days Icrmal d' 1506 91 Bent a” 1501 91 Nbrlal P1 9 651 163 lormal r1 6‘ 650 165 Bent 1'1 6‘ 652 165 Bent 11 c‘ 648 165 Bent r1 6' 655 163 sat-Bent 9 473 299 . Right Left Right Left Right -Lnft Right Left Right Left Right Left Right Left Right Left in gms. in gms. 0.1206 0.0559 0.1219 0.0577 0.1503 0.0704 0.1405 0.0674 0.1275 0.0699 0.1267 0.0710 0.1419 0.0764 0.1510 0.0805 0.1591 0.0877 0.1568 0.0861 0.1510 0.0795 . 0.1540 0.0815 0.1594 0.0756 0.1481 0.0814 0.1751 0.0875 0.1659 0.0822 Age in Femur Height of bones Ieight of ash. % Ash before igniting after igniting 46.35 47.33 46.84 48.04 54.82 56.04 53.84 53.31 _ 55.12 54.91 52.65 52.79 54.23 54.96 49.97 49.55 e weight after extraction of fat and drying. in!- 9 475 had been lactating fa. 28 days. -46- percentages of the femurs of these two animals nor is either below what would be expected for normal rats of that age. The animals which were 163 days old had been fed on the varied diet for a short time during their early life but from the age of 48 days had been placed on the control diet after which time bent-nose in the three animals became apparent. Note that no significant difference occurs between the bent and normal sibs nor is the ash percentage for any of the five lower than nomal expectancy. Female 475 had no animal with which to be compared. The ash percentage for 9 475 is a little low for an animal of that age, but this 'is due to her having been lactat- ing for 28 days. In summary, these results indicate that these bent- nosed animals were not rachitic. It is certain they were not rachitic at the time the test was made. 9 g‘he line test. Four bent-nosed and two normal animals from the ex- periment were subjected to the line test in an attempt to dis- cover whether the bent-nosed animals tended to be rachitic or not. The prinry purpose of the line test, fonnulated by Steenbock and others, was not a means by which rickets could be detected in animals, but to aid in determining the vitamin content in a food, -- usually milk. However it is convenient for giving an indication of rickets in animals. * These conclusions were drawn after consultation with Dr. 0. A. Hoppert of the Chemistry Department, Hichigan State College. .47- The method of using this test is as follows; The animals are etherised and the radius and ulna removed. Any long bone would suffice, but the radius and ulna, being most convenient, are nearly always used. Care must be taken in removing these bones not to break off the epiphysis. The dis- tal 1/5 to 1/2 of these bones .is cut from the rest of the bone and longitudinally split, then submerged in a solution of silver nitrate. The silver nitrate reacts with the calcium phosphate forming silver phosphate. In the presence of light, silver phosphate is reduced, setting free the silver. This en- ables one to distinguish between calcified areas and carti lag- inous areas as the former appear dark. A rachitic bone can be detected by the appearance of the metaphysis and the size of the end of the bone. A normal bone has a very narrow carti laginous metaphysis’ while that of a rachitic bone is quite broad due to the decalcification of the adjoining portions of the diaphysis and epiphysis. This normal metaphysis plus the decalc‘ified areas on both sides is often referred to as a rachitic metaphysis. rho end of a rachitic bone becomes much larger than normal as if to compen- sate for the strength lost by decalcification. This enlarge- ment can usually be detected by palpation. Five of tin animals, 5 bent and 2 norma1,subjected to the test ranged from 91 to 105 days of age. A fmrth bent animal was 185 days old. Had the animals ever been rachitic it shmld have shown up in the animals from 91 to 105 days of age. The animal 185 days of age was old enough that it could have recovered from the condition. -48- lhen the readings on thesebones were made, it was discovered that all animals with but one exception showed no increase in width of the metaphysis. This exception was bent 9 491 (Figure 5) and the metaphysis was not sufficiently wide to be classed as rachitic. Most of the animals showed a very slight bulging of the em of the diaphysis but this was not comparable to that of a rachitic animal. All readings were verified by Dr. C. A. Happert of the Chemistry Department. There was no difference between the appearance of the bones from the bent animals and those from the normals. .Figure 5 gives sketches of the bones of these rats and that of a standard rachitic animal, 1.9., one fed on the Steenbock rachitic ration for 51 days. Conclusions Drawn From Observations on the Effects of Nutrition. (1) Diet is a factor influencing the expression of the character bent-nose of the rat (Rattus norvegcus). *(2) x-ray photographs indicated that the bent-nosed animals were not rachitic at the time the photographs were taken. *(3) or the animals subjected to the bone ash determina- tion of the femurs, no significant difference appeared between bent-nosed animals md their normal sibs, nor were any of the animals abnormally low in percentage of ash. *(4) None of the animals subjected to the line test showed rachitic metaphyses, and no significant difference was detectable between bent-nosed and normal animals. * These three statements are indications rather than conclusions due to the paucity of animals subjected to each test. -49.. Negative of a standard rachitic rat, i.e.,one fed on Steenbock rachitic ration for 51 days. Normal 9 490 Bent 9 491 Bent 6'492 Bent F1 9 617 Normal P1 9 629 Bent P2 9 525 Fig. 5 Negatives of the line test with one standard rachitic negative for comparison. Note that these negatives show normal metaphyses with one exception which is only slightly wider than normal; also, all negatives show little or no bulging. -50- PART III HEREDITY OF THE BENT-NOSED RAT lhen one attempts to review genetic literature, he is struck by the paucity of bone abnormalities which are definitely explained on an hereditary basis. More malformations of the bone are attributed to nutrition factors than to those of heredity. It is quite probable that in.many cases both types of factors are working together to bring about the expression of the character. The work done on the flexed tail mouse by Hunt, Mixter and Permar is one of the most thorough.genetic analyses ever’made of a bone abnormality. This is an anomaly in which the caudal vertebrae are malformed and fused,causing flue tail to appear flexed in varying degrees. The condition has been found to be inherited as a recessive with modifying factors. Brown and Green (1952) made a study of hereditary variations in the skull of the rabbit. Quoting from their paper which appeared in Science, "Genetically, the importance of the observations recorded lies in the fact that a profound variation in the form.of the skull can be transmitted, unaltered, from parent to offspring; by crossing an.animal possessing a skull of this type with a normal, the original variation can be re- solved into a group of component parts which bear little or no resemblance to the original.condition, but these are inherited as distinct entities; they are differentiated into night and left sided characters and can be recombined to form the parental type as well as new types which are capable of further trans- mission.” -51- A genetic craniometric study of two species of mice, by Green.(1952), has daown that the size of the skull tends to be inherited. However Green states that a thoroughly satis- factory genetic explanation appears impossible with the data he has gathered. Another study more closely related to the bent-nose study was that reported by H. 8. Colton on the "high-brow" rat (1929). This was a brief study and it failed to demonstrate whether the character was or was not inherited. As the reader has observed, with one exception these investigators have found no definite genetic explanation for the bone anomaly with which they were working. The author re- grets that he too has found no genetic solution.flor his respec- tive problem. However, an attempt was made to determine the inherit- ance. The primary purpose of the experiment was to determine whether bent nose is inherited, and if so, how. Other phases of the situation were unexpectedly encountered. Had not the analysis been divided into so many periods, thus distributing the data into four categories, more definite discoveries con- cerning the genetics of the character might have been made. Nevertheless, the data gathered do throw some light on the solu- tion and.are well worth presenting. Technique Used in Conducting Breeding Experiments. In order to prosecute an experiment with sufficient accuracy, several precautions must be adhered to. The subse- quent rules were followed throughout the experiment. -52- (1) No animal entered the experiment without receiving a number which was marked in its ear. (2) No females were mated which.had not previously been isolated for at least twenty-one days. (3) Femles were usually isolated before parturition. In a few cases they were allowed to remain with the male, but these were instances in which no other female was in the breed- ing cage. (4) Ydung were weaned and.marked at the age of 28 days or less in all cases when the mother was remated before 21 days after weaning her progeny. (5) Thongs the date at which an animal appeared bent- ncsed was always recorded, no animals were definitely pronounced bent-nosed or straight until after they were mature, at least 120 days old, and their skulls had been removed and examined with a straightedge. A few exceptions to this statement are animals now running in the experiment, but which are definitely bent-nosed. (6) With the exception of food, which has been dis- cussed previously, all environmental factors were controlled. Presentation and Discussion of Data. Due to the changes made in food and the radical re- sults of these changes, it is necessary to divide these data into the four periods used in the study of nutritional effects. -53- Data obtained during first period. As the reader will recall, the author began the experi- ment with a number of Mr. liren's animals. These could be class- ed in the following categories: (1) F1 animals with.no bent- nosed sibs, (2) ’1 animals with bent-nosed sibs, (3) bent-nosed rats, and (4) normal.progeny of bent x bent. By Fl's is meant progeny of bent x normal. The results of the matings of these animals are given in table II. Progeny of other P1 matings are included. These P1 matings were not of rats from Mr. Wiren's stock but were of bent-nosed animals from the red-eyed-yellow stock 3: normal animals from stock unrelated to the red-eyed- yellows. The results of the matings were not very striking in this period. Only five bent-nosed animals, compared to 107 normal, were reared. The paucity of the number’ of bent-nosed animals is evidently due partly to the animals having been put on the fox-chow ration before reaching maturity. The ages of these rats when put on fox-chow ranged from 7 to 63 days. Two of the bent-nosed animals appeared in the progeny from.the mating, 1'1 (with bent sibs) 1 F1 (with bent sibs). One appear- ed in a backcross mating of ’1 d'(with no bent sibs) x bent 9, and the other two appeared in the backcross mating of F1? (with.bent sibs) x bent 6% Of the six F1 animals reared, all were normal. These data indicate that if the character were inher- ited, it followed no simple Mendelian scheme, and since no F1 animals were bent-nosed one would suspect that the character was recess ive . .54- Table II ANIMALS BORN BEFORE JANUARY‘I , 1933 Type of mating Total Normal Bent-nosed offspring offspring offspring a 9 a 9 d' 9 III (with no bent sibs) x P1 (with no bent sibs) 3 6 3 6 1'1 6' (with bent sibs) x 1'1 9 (with no bent sibs) 25 1'7 25 1'7 1’1 0" (with no bent sibs) x F1 9 (with bent sibs) 2 2 1'1 (with bent sibs) x 1'1 (with bent sibs) 5 6 3 6 2 1'1 6‘ (with no bent sibs) x Bent 9 4 3' 1 '1 6' (with bent sibs) x Bent 9 '7 3 '7 3 Dent d'x r1 9 (with bent 31b.) 11 e 10 7 1 1 P1 6' (with bent sibs) x Normal 9 (progeny of bent x bent) 6 3 6 3 1'1 latings (Bent d' x Normal 9 unrelated to bent stock) 2 4 2 4 fetal 000.000.000.000... 112 107 5 -55- Data obtained during second period. The matings made during the first period were kept breeding during the second period. A total of 191 animals -were reared during this period, but since the fox-chow sup- pressed the character these data are of no value in studying the inheritance of bent-nose. Results of this period are given in table 1:. Data obtained during third period. The most valuable data for the inheritance study were gathered during this period. These data are tazbulated in table II. Matings of the previous periods were continued and in addition were made five more F1 matings, four brother-sister matings of normal progeny from bent x bent matings, and 12 P1 matings of bent x normal animals from the bent-nose stock. The latter is not a true P1 mating as these normals might carry some of the factors for the expression of bent-nose, but not show the character as they lacked the complete necessary set- up. As the reader will recall, the author was sorely in need of bent-nosed animals, this being his reason for setting up these matings which were quite likely to produce such animals. On analyzing these data one finds several points which indicate that the character is influenced by heredity. In table II one will observe that inbreeding tends to produce more bent-nosed animals. In the 2‘7 progeny of bro ther-sister matings of the normal animals from the bent x bent matings, Table I AllIlALS REARED ON Fox-snow Type of matings Progeny .2. _2_ ’1 (with no bent sibs) x 1'1 (with no bent sibs) 41 35 ’1 6 (with no bent sibs} x Pl 9 (with bent sibs) 10 18 1'1 6‘ (nth bent sibs) x 1'1 9 (with no bent sibs) 13 12 P1 (with bent sibs) x 1'1 (with bent sibs) '7 4 1'1 6' (with no bent sibs) x bent 9 .15 10 Bent d' x P1 9 (with no bent sibs) 2 5 Bent d x 1'1 9 (with bent sibs) l 3 1'1 6‘ (with no bent sibs) x normal 9 (Progeny of bent x bent mating) 6 9 Total ................................. 191 All animals were normal. -57- Table II ANIMALS BORN AFTER MAY 5, 1935, AND FED ON VARIED DIET. Type of mating Total Normal Bent-nose % offspring offspring offspring Bent 1'1 (with no bent sibs) x . ’1 (with no bent sibs) 53 40 34 22 21 18 41 f Backcrcss 11 9 (with bent sibs) x Beat 6' 3 2 1 2 2 4O % Backcross P1 6‘ (with no bent sibs) x Bent 9 6 6 3 2 3 4 58 75 Bent-nose x Bent-nose 2 2 2 2 100 75’ 11 mating (Bent 6 x normal 9 unrelated to bent-nose 6 3 4 2 2 l 33 % stock) Normal progeny of bent-nose x bent-nose, mated inter 16 ll 4 5 12 6 6'7 % se P1 mating (Bent-nose 6 x normal 9 from bent- so 22 20 12 10 10 38.3% nose stock) TOtll eeeeeeeeeeeOO 204 111 93 ‘506‘ -53- 671 were bent. The four progeny of the bent x bent mating were all bent-nosed. Of the 12 progeny of the backcross P1 6‘ (with no bent sibs) x bent 9, 58% were bent-nosed, 41% of the 95 ’2 animals were bent-nosed, and of the 52 F1 animals from the P1 smting (bent x normals from the bent stock) 38.5% were bent-nosed, while of the nine ’1 animals from the P1 mating (bent x normals from.unrelated stock) 33% were bent-nosed. In only three classes are the numbers of individuals great enough to give frequency percentages of statistical value. These were: (1) progeny whose parents were normals from bent x bent, mated inter se, (2) progeny of bent dB‘x normal 99 from.the bent stock, and (3) Fz's from.F1's (with no bent sflbs; x 11's (with no bent sibs). Between the first and second class, a frequency of bent percentage difference of 28.51 '7.61 exists, while between the first and third class there is a difference in frequency of bent percentages of 261t 6.99. These are both significant, but the difference between classes two and three is 2.3* 5.68 which is not statistically significant. (Table III.) The first two differences would indicate that there is an apparent decrease in the percentage of bent-nosed animals as the concentration of bent-nose declines. It is true that the animals were not fed on a con- trolled diet, which mig1t account for one animal developing bent-nose when his sib was normal, since there was a chance for some rats to select certain ingredients and to reject others.in the imperfectly mixed ration; but it is hardly prdbable, considering the group as a whole, that one would -59- Table XII PERCENTAGE OF BERT-ROSED PROGERY WITH DECREASING PROPORTION OF BERT ANCESTRY Parent Number of % bent among proagg; nroagg_ Bent x bent 4 100 lcrmals from bent x bent, inter se 2'7 671: 6.10 11 2 (with bent sibs) x bent o’ 5 4o ’1 o’ (with no bent sibs) x bent 9 12 58 Bent <51 x normal 9 from bent stock 52 38.5:t4.55 r (with no bent sibs) x P (with 1 no bent sibs) 1 95 41 :1: 3.41 Bent c” x normal 2 from non-bent stock 9 33 -60- find such.marked differences in the percentage of bent-nosed animals in the different classes were due wholly to selection of food. It would be possible for animals to inherit certain tastes which.the inbreeding brougat out, but still the charac- ter would be indirectly influenced by heredity. In short, if the bent-nosed condition is due wholly to dietary influences, why do not all the types of matings produce substantially the same percentage of bent animals irrespective of ancestry? If the character is hereditary,.it:must be influenced by dominant factors as bent-nose Pl animals appear. One could not conclude that it was or was not sex-linked since, due to the shifting from.one period to another, data on reciprocal crosses were not obtained. In all, only 204 animals were reared in this period; and since these were divided into so many different categories, the numbers from any one type of mating are not great enough to justify one's drawing definite conclusions. ~The percentage differences may have been due exclusively to non-genetic causes operating within.small numbers. Data obtained during fourth period. The data of the fourth period were Obtained under a condition in which every factor was controlled with the except- ion of heredity. Any variation in the frequency of bent-nose in these animals could not be attributed to food. If heredity were not a factor in the production of the'bent-nosed character, one would expect all animals to be either bent-nosed if the food -61- was sufficiently deficient to cause the character, or normal if the food was of such a constitution to prevent the development of the anomaly. Three animals from a total of 143 developed bent-nose during this period. (See table XIII for data.) Two of the bent-nosed animals were derived from bent-nose x bent- nose crosses, while the third was from a backcross. None of the P1 or P2 animals deveIOped the character. It would have been necessary to have had more bent-nosed animals appear to prove tlmt the character was influenced by heredity. However, only on the assumption that hereditary influences exist can the appearance of the three bent-nosed'animals be explained. It is quite possible that this controlled diet was much more nearly balanced than the combination fed during the third period. Though this contained the same ingredients of the varied diet, the preportions were not the same since those of the latter were not known. Probably a higner frequency of bent-nosed animals would have appeared during this fourth period had the diet constituents been in the same preportions as those in the preceding period. Four females and two males were imported from the Iistar Institute October 13, 1933, and were mated to secure some control animals. From these control matings 64 animals were reared, all of which were fed on the controlled diet. None of these animals .has shown the bent-nosed character. However, as previously cited, only 13 are older than 120 days, though the remainder are over 63 days of age. Since none of these animals .has been etherised, only a superficial examina- tion has been made. -52- Tablc XIII ANIIALS BORN AFTER NOVEMBER 18, 1935, FED ON CONTROLLED DIET. Type of mating Total Normal Bent-nosed offspring offspring offspring 6' 9 6' 9 6' 9 ’1 (with no bent sibs) x P1 (with no bent sibs) . 21 29 21 29 Backoross F1 x Bent 24 33 24 32 l Bent-nose x Bout-nose 11 8 9 8 2 P1 mating (bent d‘ 1 normal 9 unrelated to bent-nose stock) 11 6 11 6 Tflal ....OOOOOOOOOOOOOOOO 143 140 3 Control matings (normal x normal 'istar animals) 37 27 37 27 64 64 Another aspect of the problem which was presented during the anatomical study, but which gives evidence of heredity, was the specifity with which.the factors causing bent-nose manifested themselves. As the reader will recall, the condition is normally accompanied by no other bone ab- normality. Though this does not prove that bent-nose is in- herited it is more characteristic of the action of hereditary factors while nutritional deficiencies alone usually have a more generalized effect. Summary of Heredity Study. until more complete data have been obtained, defin- ite conclusions will not be drawn as to whether the bent-nosed character is inherited or not. The following facts are evi- dence that bent-nose is inherited. (l) The character appeared in the red-eyed-yellow stock and not in the three stocks of the colony that were un- related to the red-eyed-yellows, though all were subjected to similar enyironment. (2) It appears to be possible to breed for high frequency of occurrence of the bent-nosed character. (3) Three animals developed bent-nose in a total of 143 animals when all factors but heredity were controlled. (4) The factors causing bent-nose are specific in their manifestation. Normally no other structural anomaly accompanies the bent-nosed character. GENERAL DISCUSSION Though the data are not conclusive, it appears that bent-nose is the resultant of the interaction of several causative agents including‘bcth environmental and heredity factors. It seems that a definite genetic constitution is necessary for the appearance of the anomaly,but this can be manifested only when the diet is deficient. It has been definitely proved that food is a factor. Prom matings which produced 191 animals, all normal, on fox- chow, breeding with a varied diet yielded 117 of which 52, or 44$, developed bent-nose. [any facts indicate that the character is genetic. The causative factors are specific in their manifestation; inbreeding tends to increase the frequency of bent-nosed animals; the condition was prevalent in only one strain of the colony; and when all enyironmental factors are controlled three animals developed the anomaly. All of these tend to prove that the character is inherited, and when a thorough analysis has been completed, heredity may be found to be quite as important as diet. The most nearly conclusive evidence of bent-nose being hereditary is the apparent increase in the percentage of bent- nosed progeny with.increasing proportion of bent ancestry. (Table III.) If the character were genetic, one would expect a higher frequency of bents among the progeny of normals from bent x bent, meted inter se, than among the 32's from.P1's with no bent sibs 1 11's with no bent sibs. Of the first class 67% -55- 'were bent, while only 41% of the Pz's were. This is a statistic- ally significant difference. The frequency of bent-nose among the progeny of bent dB'x normal 99 from.the bent stock was 38.5% which is also significantly less than that of the progeny of normals from bent x bent, mated inter as. This difference is of less value than the former, due to the possible variability in the genotypes of the normal females from the bent stock. One would expect the character to be rachitic in nature. However, the tests have indicated that the animals are not rachitic. Those examined did not show a rachitic metaphysis, an abnormally low percentage of bone ash content, nor other structural anomalies usually present in rachitic animals. How- ever, as yet these tests are not conclusive. The problem is not solved; only some of its phases have been revealed. Before the genetics can be analyzed, a controlled diet which will give a high frequency of bent-nosed animals must be formulated. A homozygous bent-nosed strain that could be crossed with.normals should be developed by inbreeding. Then with a controlled and deficient diet and a homozygous strain it should be possible to discover many facts on the inheritance of the character. Though it has been proved that food is a factor, its causative elements are unknown. These should be determined. The study of rickets should be continued.more extensively to determine whether there is some bone deficiency just at the time bent-nose is developing. -55- GENERAL CONCLUSIONS (1) Bent-nose, a character of the rat, has its expression centered on the rostrum of the skull. (2) Diet is a factor in determining whether or not animals will be bent-nosed. (3) Though.the data are inadequate to prove definitely that bent-nose is inherited, mmny facts strong- ly indicate the presence of genetic factors. -67.. BIBLIOGRAPHY Blunt, Katharine, Cowan, Ruth, 1931 'Ultra-violet Light and Vitamin D in Nutrition” The University of Chicago Press Colton, H. 3., 1929 "High.brow' Albino Rats‘I Journal of Heredity, Vol. 20, pp. 225-227 Gray, Henry, 1934 "Anatomy of the Human Body" Twenty-second edition by warren H. Lewis pp. 124-195 '"The Skull", Lea and Febiger Green, 0. 7., 1932 "A Genetic Oraniometric Study of Two Species of Nice and Their Hybrids" Journal of experimental zoology, Vol. 63, pp. 533-551 Green, Harry 8. N., Brown, wade H., November 4, 1932 "Hereditary Variations in the Skull of the Rabbit" Science, Vol. 76, pp. 421-422 Howell, A. Brazier, 1926 “Anatomy of the Wood Rat” Chapter VIII pp. 110-172.”Osteologv" The lilliams and wilkino company Hunt, Harrison H., Nilter, Russell, and Permar, Dorothy, July 1933 ”Plexed Tail in the House, Nus1musculus' Genetics, Vol. 18, pp. 335-366 -53- BIBLIOGRAPHY Cont inued Hunt, H. H., 1925 "A Laboratory nanual of the Anatomy of the Rat" pp. 6-37 "The Skeletal System? The Iaolillan.Company _ Hrdllo’ka, 1158, 1920 "Anthropometry" The Wistar Institute of Anatomy and Biology lcCollum, E. V., Simmonds, Nina, 1929 "The Never Knowledge of Nutrition" ”The Use of Foods for the Preservation of Vitality 'and sea1th"‘ Chapter III pp. 289-332 "Experimental Rickets" The lacMillan Company _ Hills, Frederick Cecil, 1930 "Statistical Methais Applied to Economics and Business" Henry Holt and Company Sherman, Henry 0., 1928 "Chemistry of Food.and.nutrition" Chapter xVII pp. 471-492 "The Antirsehitic Vitamin and the Prevention of Rickets" The lacMillan.Company Shohl, Alfred T., Brown, Helen B., Chapman, Edna 3., Rose, Catherine 3., Saurvein, issther H., May,1933 ”The EValuation of the Phosphorus Deficiency of the Ricknts-producing Diet” Journal of Nutrition, Vol. 6 pp. 271-283 -69.. BIBLIOGRAPHY Continued Sure, Barnett, 1933 I'The Vitamins in Health and Disease” Chapter v pp. 83-114 ”Vitamin D" The Williams and iilkins Company -70- EXPLANATION OF PLATES Platest and II are photographs of the bent-nosed character. Plate I is an external view while II is a dorsal View of cleaned bent skulls. Plates III and IV demonstrate apparatus and tech- nique used in measuring skulls. The remainder of the plates are X-ray photographs, the first (plate V) being a normml animal while the remain- ing eight are bent-nosed animals. These photographs had two purposes, namely: (1) anatomical study, (2) rachitic test. -71- Plate I External appearance of a bent-nosed animal. Adult animal with nose bent to the right. -72- Plate II Skulls showing the bent-nosed condition. Female 6 normal. Female 7, Female 9 and Female 11 bent. ‘ The absence of the zygomatic bone is not an anomaly as in each case it was lost while the skull was being Cleaned. -73- Plate III Strain gage mounted with points for making all skull measurements except the length. Iicrometer to the right used for measuring the length of the normal skulls. -74 Plate IV Demonstrating:method used for measuring the angle of bend in the skulls. Plate V An X-ray photograph of normal Male 1001 from the control stock. Age 108 days. Plate VI An x-ray photograph of bent Male 18, a progeny of a brother-sister mating of normal animals derived from a bent x bent mating. Age 217 days. -77- I . I - 1 1“ is ch) .( \(hmrf’c’é .n... .— ————V Plate VII An X-ray photograph of bent F2 Female 351, Age 167 days. -73- L Plate VIII An x-ray photograph of Bent F2 Male 375 Age 167 days. -79- I Plate IX An X-ray photograph of bent F2 Male 369, Age 167 days. -80- I 1 Plate X An X-ray photograph or bent Male 58, a progeny of a brother-sister mating of normal animals derived from a bent x bent mating. Age 217 days. -31- Plate XI An X-ray photograph or bent Female 108, a progeny of a brother-sister mating of normal animals derived from a bent x bent mating. Age 164 days. I6; - A“ q . A Plate XII An X-ray photograph of bent Hale BB, a progeny of a brother-sister mating of normal animals derived from.a bent x bent mating. Age 204 days. -83- ...-o“.- Plate XIII An X-ray photograph of bent Male 193, a progeny of a brother-sister mating of normal animals derived from a bent x bent mating. Age 193 days. ROOM USE ONLY MICHIGAN STATE UNIVERSITY LIBRARIES 12 3 93 03015 2185