V itam in D Studies and Rickets / -By H. ERNEST BECHTEL <1 A THESIS 1 ’resen ted to the G raduate School of M ichigan State C ollege of Agriculture and A p p lied Science in Partial F ulfillm ent of Requirements for the D egree o f D octor o f Philosophy Chem istry Departm ent East Lansing, M ichigan 1935 ProQuest Number: 10008490 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest ProQuest 10008490 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 R eprinted from T h e J o u r n a l o p N u t r i t i o n Vol. 11, No. 6 and Supplement, June, 1936 A STUDY OF THE SEASONAL VARIATION OF VITAMIN D IN NORMAL COW’S M IL K 1 H. ERNEST BECHTEL AND C. A. HOPPERT Departm ent of Chemistry, Michigan State College, E ast Lansing, Michigan T H R E E FIGU RES (Received for publication February 7, 1936) In recent years a great deal of attention has been given to the importance of vitamin D in the human dietary and particularly to the desirability and means of enhancing the milk supply in this factor. The methods by which the latter has been achieved are well known and relatively little thought is now given to the antirachitic potency of milk produced under ordinary farming conditions. Although many studies have been made of the vitamin D content of cow’s milk, none of them has been of a comprehensive nature. Table 1 sum­ marizes the results of some of these investigations. Similar trends in variations of the vitamin D potency of milk are indicated by the work of other investigators. Luce ( ’24) found milk from pastured cows definitely richer in vita­ min D than that from stall-fed cows kept in the dark. Luce concluded that the concentration of the antirachitic factor in milk depended on the ration and possibly also on the degree of illumination received by the cow. Chick and Roscoe ( ’26) found 0.25 gm. of milk fat from pastured cows equal to 0.60 gm. milk fat from cows fed green feed in a dark stall. Ex­ posure of the cow to outdoor light without a change in ration resulted in a twofold increase in the vitamin D content of 1 The data in this paper -were taken from the thesis presented by H. Ernest Beehtel to the faculty of the Graduate School of Michigan State College in 1935 in partial fulfillment of the requirements for the degree o f doctor of philosophy. 537 T H E JO U R N A L, OP N U T R IT IO N , VOL. 1 1 , N O . 6 AN D S U P P L E M E N T 538 H . E. BECHTEL AND C. A . H O P P E B T the milk fat. Dutcher and Honeywell ( ’27) examined some Kansas butter samples and found that milk fa t from cows exposed to sunshine was superior in vitamin D potency to fat produced by cows fed in the dark. These and other studies which might be mentioned indicate in a general way the variations that occur in the vitamin D potency of milk and the factors which contribute to this varia­ bility. However, most of the work done has been of a rather fragmentary nature so that it seemed desirable to make a more extended study of the subject. TABLE 1 V I T A M I N D C O N T E N T O F N O R M A L M IL K U N IT S P E R QUART SOURCE O F DATA U.S.P.* Steenbock 21.3 36.5 7.9 13.5 Mitchell and associates (Pennsylvania) ( ’32) Summer milk 16.2 6.0 Krauss (Ohio) ('33) Winter milk Summer milk 7.6 16.2 2.8 6.0 Russell (New Jersey) ( ’33) Summer milk Always less than 43.2 16.0 Steenbock and associates (Wisconsin) ( ’30) June 1925 September 1925 1 The values in this column were obtained by multiplying the number of Steenbock units by 2.7. MATERIALS A N D METHODS This study was begun in 1932, the milk being derived from various sources. Milk from the Holstein and Guernsey herds at Michigan State College was assayed monthly over a period of 2 years. Only the higher producers, namely, cows that were being milked three times daily were used, the average number of Holstein and Guernsey cows being fourteen and eight, respectively, for the 2-year period. From these cows 100-pound portions of composite 24-hour samples were saved monthly. Monthly samples of a similar nature were derived from a few of the highest producing cows in the Holstein herd of the Michigan Experiment Station. These cows were kept on a ration of alfalfa hay, silage and corn. VA K IA TIO N OF V IT A M IN D I N M IL K 539 From July 1933 to July 1934 monthly samples of Michigan State College Creamery butter, made from milk produced by local Michigan dairymen, were also put aside for vitamin D assays. Approximately fifteen herds, consisting largely of grade Holstein cows were represented in this group of samples. In the case of the milk a small portion was used for a fat determination, the remainder being run through a cream separator. The butter obtained by churning the cream was packed in paper cartons and stored at 0°F. until the samples were to be assayed. At this time the butter was heated on a steam bath for about an hour and the relatively pure milk fat upon which the assays were conducted was suctioned off. Except when needed for assay, the fats were kept at 0°F. The vitamin D determinations were carried out by the curative feeding technic, several changes being made in the official procedure. The Steenbock basal ration was slightly modified to obtain more consistent and somewhat better growth of the rats during the preliminary period. The rachitogenic diet used throughout this study was composed of the following: P e r cen t Yellow cornmeal 38.0 Oatmeal 37.5 Wheat gluten 20.0 Calcium carbonate 3.0 Sodium chloride 1.0 Yeast powder 0.5 Instead of feeding the fat as a daily supplement during the first 8 days of the test period, the entire amount was mixed with 40 gm. of the basal diet. This mixture was found to be consumed in 6 to 8 days after which the basal diet was fed to finish the 10-day period. Control rats receiving 29 mg. of Official Reference Oil equivalent to 2.7 U.S.P. units were used for comparison. The usual staining technic was applied to the radii and ulnae. In carrying out the assays a preliminary test was made to determine the approximate vitamin D content of the vari­ 540 H . E. BECHTEL AND C. A . H O P P E R T ous samples. The confirmatory tests were then conducted at three levels using three to five rats at each level. ^ It became apparent early in this investigation that fats of low potency could not be assayed because of the limited capacity of the rats to consume fat. Amounts up to 6 and 8 gm. were consumed fairly consistently. However, when the dosage was increased to 10 gm. approximately only half of the test animals consumed the fat-basal diet mixture in 8 days. Attempts were therefore made to effect a concentration of the vitamin D so that fats of lower potency might be assayed. Although the work of Kon and Booth ( ’34) indicated that at least a part of the vitamin I) of butter fat was unstable and could not be recovered quantitatively in the non-saponifiable matter, their method as well as several modifications were given a trial. Sometimes the recovery of vitam in D was quantitative, but more often it was not, so that this method of concentration was abandoned. Inasmuch as the concentration of vitamin D in cod liver oil may be accomplished by extraction of the oil with alcohol, this method was next tried and proved to be satisfactory. The fats of low potency were therefore treated in the following manner: 100 gm. of melted milk fat was placed in a separa­ tory funnel previously warmed in a 37 °C. oven and 100 cc. of hot ethyl alcohol (95 per cent) was added. The mixture was then shaken fairly vigorously and the funnel placed into the 37° C. oven until the layers had separated. The fa t was then drawn off into a warm beaker and the alcohol layer into a 500 cc. volumetric flask. The fat was returned to the sepa­ ratory funnel and the beaker rinsed with 50 cc. of hot alcohol which was then added to the fat. The mixture was again shaken and the layers allowed to separate in the oven. The separations were made as before and three additional ex­ tractions carried out with 50 cc. portions of hot alcohol. B y this process approximately 20 per cent of the fat was removed and this fat contained all of the vitamin D. The combined extracts were brought to a volume of 500 cc. with ether in order that the fat would be kept in solution. It was observed V A R IA T IO N OF V IT A M IN D I N 541 M IL K that the antirachitic value was retained for at least 2 months when the extract was stored at 0°C. Aliquots of this solution were poured on 40 gm. portions of the rachitogenic diet in evaporating dishes and the ether and alcohol allowed to evaporate spontaneously. These mixtures were then fed in the usual manner. TA BLE 2 Antirachitic potency of milk f a t obtained from the Holstein h erd1 DATE F A T -C O N T A IN ­ IN G 1 U .S .P . V IT A M IN D U N IT AVERAGE D AILY PR O D U C T IO N P E R COW FAT IN M IL K V IT A M IN D P E R QUART O F M IL K Fat V itam in D U.S.P. gm. gm. U.S.P. per cent 1.5 1.1 1.1 3.3 817 717 862 748 545 701 784 227 3.26 2.88 3.53 2.78 — — — — — — — — — — — — January February March April May June 5.6 9.3 3.7 3.7 3.0 826 875 835 748 730 148 94 226 202 243 3.15 3.26 4.6 3.1 6.7 5.9 7.0 1.7 1.1 2.5 2.2 2.6 — — — — — — July August September October November December 1.5 1.9 1.5 3.7 7.4 776 862 780 690 753 517 454 520 186 102 3.20 3.28 2.98 2.96 3.19 16.6 14.9 15.5 5.5 3.2 6.1 5.5 5.7 2.0 1.2 — — — — — — — 7.4 7.4 4.4 3.0 — 844 939 871 898 — 114 127 198 299 — 3.17 3.29 3.06 3.31 — 3.6 4.2 6.1 9.9 — St.2 AVERAGE D A IL Y A M O U N T OF S U N S H IN E hours 3 1932 July August September October November December 17.8 20.2 27.7 6.3 6.6 7.5 10.3 2.3 11.5 10.0 7.8 3.3 4.0 3.0 1933 2.97 2.93 2.89 4.1 7.1 4.4 6.8 8.5 12.7 12.8 10.8 7.1 6.0 2.0 1.4 1934 January February March April May 1.3 1.6 2.3 3.7 2.3 5.9 5.0 6.0 11.3 1 Average of fourteen cows per month. 2 Steenbock. ’ Average amount of available sunshine according to the East Lansing Weather Bureau. H . E. BECHTEL AND 542 C. A . H O P P E R T The above method permitted practically complete recovery of the vitamin D from samples of milk fat of which 2 to 10 gm. had to be fed of the original fat to get the typical narrow continuous line of calcification. Inasmuch as there was no way of checking the fats of lower potency directly, it had to be assumed that the method was also satisfactory for such TABLE 3 Anti/raohitic potency of milk fa t obtained from the Guernsey h e rd 1 f a t -c o n t a i n DATE ­ 1 U .S .P . V IT A M IN D U N IT in g AVERAGE D A IL Y PR O D U C T IO N P E R COW FAT IN M IL K V IT A M IN D P E R Q UART O F M IL K U .S.P. St.2 AVERAGE D A IL Y A M OUNT OF S U N S H IN E Pat Vitamin D gm. gm. U.S.P. 1.1 1.1 1.3 3.0 3.7 3.7 649 685 708 699 708 930 590 623 545 233 191 251 4.93 4 .86 5.15 4 .52 ' 4 .02 4.76 4 3 .8 43.1 38 .7 14.7 10.6 1 2.6 1 6.2 16 .0 1 4.3 5.4 3.9 January February March April May June 4 .4 9.3 3.7 3.0 3.0 907 835 762 658 694 206 90 206 219 231 4.42 4 .57 4.33 4.34 4.42 9.8 4.8 11.4 14.1 14.4 3.6 1.8 4 .2 5.2 5.3 — — — — — — July August September October November December 1.5 1,9 1.9 2.6 3.7 — 744 853 694 721 912 — 496 449 365 277 246 4.43 5.09 4.61 4.76 4.86 28.8 26.2 23.7 17.9 12.8 1 0.7 9.7 — — — — 1 2.8 1 0.8 7.1 6.0 2.0 1.4 2.2 2.9 4 .4 5.9 2.3 5.9 5.0 6.0 11.3 per cent hours 3 1932 July August September October November December 4 .7 1 1.5 10.0 7.8 3.3 4 .0 3.0 00 00 1933 6.6 4 .7 4.1 7.1 4 .4 6.8 8.5 1 2.7 1934 January — February 7.4 March 5.6 April 3.7 May 3.0 1 * ____ 1 Average of eight cows 1 Steenbock. — — _ 866 835 826 871 117 149 223 290 4.44 4.50 4.52 4.90 5.9 7.8 11.9 15.9 per month. 3 Average amount of available sunshine according to the E ast Lansing Weather T A BLE 4 Antirachitic potency of milJc fa t obtained from the experiment station h erd 1 FAT C O N T A i: 1 TJ.S.P. V II D U N IT lb. gm . gm . U.S.P. p e r ce n t 24.2 26.3 26.4 23.1 26.0 24.9 — — 5.5 25.9 20.9 24.0 15.1 13.8 8.3 4.4 3.5 4.1 1.3 1.5 1.5 2.6 3.7 3.7 544 617 581 508 662 435 418 411 387 195 179 118 2.30 2.64 3.18 3.34 4.02 3.29 ZM MJ U .S .P . CS & E* 55 DAILY AM OU S U N S H IN E Corn lb. V IT A M IN D P E R QUART M IL K Vitamin D Silicon | Alfalfa Ph lb. DATE 1933 July August September October November December AVERAGE D A IL Y PK O DUO TIO N PER, OOW o£ £a AVERAGE D A IL Y FE E D IN T A K E m h o u rs 3 17.3 17.2 20.7 12.5 10.6 8.7 12.8 10.8 7.1 6.0 2.0 1.4 6.4 6.4 7.7 4.6 3.9 3.2 1934 J anuary 2.3 5.9 February 26.1 26.0 74 3.40 0.3 4.4 7.5 327 2.8 5.0 21.2 121 3.73 March 3.2 4.4 531 8.3 3.1 19.7 2.84 6.0 7.5 2.8 April 23.8 535 13.0 3.7 145 9.3 11.3 13.9 26.0 2.2 794 3.13 5.1 3.6 12.6 361 May 1 Average of five cows per month. 2 Steenbock. 3 Average amount of available sunshine according to the East Lansing Weather Bureau. TA BLE 5 Antirachitic potency of creamery milk f a t 1 V IT A M IN D P E R Q UART OF M IL K F A T C O N T A IN IN G 1 U .S .P . V IT A M IN D U N IT FA T I N M IL K gm . p e r cen t 1933 July August September October November December 1.3 1.9 1.3 2.6 4.4 3.0 3.5 3.5 3.5 3.5 3.5 3.5 26.3 18.0 26.3 13.1 7.8 11.4 1934 January February March April May June — 3.7 — — 3.0 1.9 — 3.5 — — 3.5 3.5 9.2 — — 11.4 18.0 DATE U .S .P . Steenbock AVERAGE D A IL Y A M O U N T OF S U N S H IN E . h o u rs 2 9.7 6.7 9.7 4.9 2.9 4.2 12.8 10.8 7.1 6.0 2.0 1.4 — 3.4 2.3 5.9 5.0 6.0 11.3 11.8 — — 4.2 6.7 1 Average of twelve herds of cows per month. 2Average amount of available sunshine according to the East Lansing Weather Bureau. 543 CO 00 jteth jo +* Q) 2•H CO I n •3 C Q, O o in o co w w (3HTW jo qjtynfo j a i ) sq iu fl a n-pureq-FA M ' S ’Il m CO o> *qi St <8 M h co CM o> CO r— I 05 r -i milk from the Holstein and Guernsey herds, and available sunshine. milk from the Experiment Station herd and M. S. 0. Creamery, and available sunshine. milk fat from Holstein and Guernsey herds, and available sunshine. strpqsuns s jnog jo jaqnm.ii ©Sujsav 3 ie> o Antirachitic potency of Antirachitic potency of Antirachitic potency of H . E. BECHTEL AND Fig. 1 Fig. 2 Fig. 3 544 C. A . H O P P E R T H 05 V A R IA T IO N OF V IT A M IN D IN M IL K 545 fats. Quantitative recovery was also obtained when a definite amount of vitamin D from the official reference oil was added to milk fat and subjected to hot alcohol extraction. The results of this study of the seasonal variation in the vitamin D content of cows’ milk are presented in tables 2, 3, 4 and 5 and portions of the data are shown graphically in figures 1, 2 and 3. The data include not only the results of the bioassays but also the average daily production of milk fat and the number of vitamin D units in the milk fat. The results were also calculated in terms of U.S.P. units per quart and these values are presented in figures 1 and 2 with the average daily hours of sunshine available each month. To simplify the comparison of these results with those given in the older literature the antirachitic potency is also expressed in terms of Steenbock units. Inasmuch as exposure of the cows to sunlight and the in­ gestion of sun cured roughages are two important factors which influence the vitamin D potency of the milk, a brief reference to the general management of the several dairy herds is appropriate. The main Holstein and Guernsey herds of the college were kept under parallel conditions at all times. Prom May to September, inclusive, these animals were pastured an average of 8 hours daily and received no hay or corn silage. During* October they were pastured an aver­ age of 5 hours daily and received about 1 pound of hay per 100 pounds of body weight. Prom November to April, in­ clusive, the animals were put out doors in dry lot for an average of 2 hours daily. During this period in 1932-1933 they received besides their allowance of grain an average of approximately 2 pounds of hay per 100 pounds of body weight, no corn silage being included. During the correspondingperiod in 1933-1934 the animals received f pound of hay and 3 pounds of corn silage per 100 pounds of body weight in addition to grain. The average weight of the Guernsey cows was 1150 and that of the Holsteins 1400 pounds. The Holstein cows in the experiment station herd were out of doors in dry lot an average of 7 hours daily from May 546 H . E. BECHTEL AND C. A . H 0 P P E R T to September, inclusive, and about 2 hours daily during the other months. These cows were kept on a ration of alfalfa hay, corn silage and corn as shown in table 4. The general management of the local Michigan dairy herds which served as the source of the college creamery butter samples was typical of that practiced in this state. The cows were fed chiefly home grown feeds consisting largely of alfalfa and cereal grains and were usually pastured as early and as late as conditions permitted. They were probably exposed to sunshine for a longer time than the cows in the college herds. Regarding the assaying of the various samples of butter, there was a considerable interval between the time the samples were obtained and the time the bioassays were made. This delay was due chiefly to the fact that a satisfactory method had to be developed before the samples of low vita­ min D potency could be assayed. However, there appeared to be no danger of a loss of antirachitic potency because some of the older samples were assayed 30 months after the first test was completed, the results indicating that vitamin D in milk fat is stable for at least 30 months when the samples are stored at 0 °F . in the dark. Practically all of the results given in tables 2, 3, 4 and 5 were obtained by using the alcohol extraction method, al­ though most of the summer samples and a few of the more potent winter samples were also assayed by feeding the original fat. d is c u s s io n In this investigation two assumptions were made which appear to be justifiable. It was assumed that all of the anti­ rachitic potency of cows’ milk is present in the milk fat and that there was no significant loss in potency incidental to the separating and churning of the cream. The standard curative feeding technic was selected for the bioassays because this method has a number of definite ad­ vantages over the prophylactic procedure. Besides the fact hat the former is much more widely used, it permits the VA R IA TIO N OF V IT A M IN D IN M IL K 547 feeding of relatively large amounts of fat without interfering with the test itself. In the prophylactic method the addition of vitamin D free fat to the basal rachitogenic diet will of itself cause a definite increase in the ash content of the bones, the increase depending on the amount of fat added. In this connection the slight modification of the rachitogenic diet seems justified because the rats attained a slightly larger size at the end of the preliminary period and had somewhat better appetites. This permitted the feeding of larger amounts of fat which was necessary in the case of the samples of lower potency. Nevertheless there were limitations in the capacity of the rachitic rats to consume fat and this necessitated con­ centration of the vitamin D. The alcohol extraction method described above seemed to solve this difficulty. Inasmuch as Kon and Booth ( ’34) had felt that the vitamin D in winter milk might be different from that in summer milk because of the difference in stability to saponification, some of the more potent winter samples were assayed both by feed­ ing the original fat and an equivalent amount of the extract. Although many of the rats failed to consume the larger doses of fat during the first 8 days of the experimental period, a sufficiently large number was used so that an assay at the 10-gm. level was made possible. The results indicated that there was no apparent loss in vitamin D in making the alcohol extractions of the winter samples tested, and served as the basis for the assumption that the assays of fats of still lower vitamin D potency by this method of concentration might be reliable. The results obtained demonstrate that milk produced by cows managed under practical farming conditions varied as much as 900 per cent in antirachitic potency, reaching a maxi­ mum from June to September and beginning with October, declining rapidly to a minimum which usually occurred in February. From the assays made on the milk fats it was calculated that the maximal potency of the milks examined in this study was 43.8 U.S.P. units per quart. Values of 20 to 30 units per quart were not uncommon during the summer 548 H . E. BECH TEL AND C. A . H O P P E E T months whereas values of 8 units and less were frequently observed during the winter months. These results in a gen­ eral way corroborate those of other investigators. Regarding the factors which contribute to the variability in the vitamin D content of milk, the amount of exposure of the cows to sunlight probably plays the major role. This is strikingly indicated by the excellent correlation between the vitamin D potency of the milk and the amount of available sunshine as shown in figures 1, 2 and 3. Undoubtedly even better correlation might have been obtained if a record had been kept of the hours of actual exposure to sunlight as well as of the ultraviolet intensity of the sunlight. The lack of agreement during February is to be explained on this basis. It follows from the above that the vitamin D contained in ordinary dairy feeds, particularly roughages and silage, how­ ever important this source may be to the general well being and productiveness of the dairy cow, contributes relatively little to the vitamin D content of the milk. Furthermore the rapid drop in the antirachitic potency of milk which follows the decrease in exposure of the cows to sunlight suggests that under ordinary conditions of management and feeding the dairy cow has practically no opportunity to build up a reserve of vitamin D during lactation. In comparing the Holstein and Guernsey samples as shown in tables 2 and 3 it is interesting to note that there was little difference in the antirachitic potency of the milk fat. How­ ever, because of the higher per cent of fat in the milk of the latter breed, the calculated vitamin D content of the milk was greater. V A R IA T IO N OF V IT A M IN D I N M IL K 549 SUMMARY 1. A method is presented for the concentration of the anti­ rachitic factors in milk fat thus making possible the biological assay of fats of low potency. 2. The monthly assay of milk fats from several sources over a period of 2 years shows that milk may vary as much as 900 per cent in antirachitic potency. Highest values were obtained during July, August or September and lowest usu­ ally in February. Vitamin D values ranging from 4.8 to 43.8 IJ.S.P. units per quart of milk were observed in the case of Guernsey milk whereas the extreme values for Holstein milk were 3.1 to 27.7 U.S.P. units per quart. 3. The close correlation between the antirachitic potency of milk and the amount of available sunshine indicates that the exposure of cows to sunlight is the major factor contributing to the vitamin D content of milk. 4. Apparently the cow has little or no opportunity to store vitamin D during lactation under ordinary dairy management conditions. LITERATURE CITED M. H. R o sc o e 1 9 2 6 Influence o f diet and sunlight u p o n the amount of vitamin A and vitamin D in the milk afforded by a cow. Biochem. J., vol. 20, p. 632. D u t c h e r , R. A. a n d H. H o n e y w e l l 1927 Feeding experiments with rats using butters furnished by Dr. J. S. Hughes of Kansas. Penna. Agr. Exp. Sta. 40th Am. Rep. Bui. 218, p. 4. K o n , S . K. a n d R. G. B o o t h 1934 The vitamin D activity of butter. III. An attempt to elucidate the nature of the labile factor in butter anti­ rachitic for the rat. The antirachitic potency of lard, olive oil, egg oil and the fatty acids of butters and lard. Biochem. J., vol. 28, p. 121. K r a t jss , W . E. 1933 Personal communication. L u c e , E. M. 1924 The influence of diet aud sunlight upon the growth-promoting and antirachitic properties of the milk afforded by a cow. Biochem. J., vol. 18, p. 716 and p. 1279. M it c h e l l , J. McK., J. E i m a n , D. V . W h i p p l e a n d J. S t o k e s 1932 The pro­ tective value for infants of various types of vitamin D fortified milk. Am. J. Pub. Health, vol. 22, p. 1220. R u s s e l l , W. C. 1933 Increasing the vitamin D content of milk. N. J. Agr. Exp. Sta. Circ. 285. Ch i c k , H. and S t e e n b o c k , H -, E . B . H a r t , B . M . R i i s i n g , C. A . H o p p e r t a n d S . B a s h e r o w 1930 Fat-soluble vitamins X X V III. The antirachitic value of cows’ milk as modified by exposure of the cow to sunlight and to radiations from a quartz mercury vapor lamp. J. Biol. Chem., vol. 87, p. 103. THE ANTIRACHITIC ACTIVITY OF VARIOUS PARTS OF THE CORN PLANT AT THE TIME OF ENSILING H. E R N E ST B EC H T E L A N D C. A. H O P PE R T SEC T IO N S OF DAIR Y H U SB A N D R Y A N D CHEM ISTRY P rev io u s w o rk a t this S ta tio n d em o n strated th a t corn silage has a n tira c h itic p ro p e rtie s w hen fed to dairy calves, and to ra ts (1). The p re se n t re p o rt is a co n tin u atio n of this w ork, and is concerned w ith the v itam in D c o n te n t of several p a rts of the corn p lan t a t th e tim e of ensiling'. As indicated elsew here (1), corn is usually h arv ested for silage when som e p a rts of th e p lan t have becom e d ry in th e field, w hile o th er p o r­ tio n s of th e p lan t rem ain green. T he available d ata su g g est th a t corn silage m ay derive its a n tira c h itic p ro p erties from th e sun-dried p o r­ tions of the corn p lan t w hich are p re sen t at th e tim e of ensiling. Experimental In 1933, several p a rts of th e corn plant w ere collected from a field of corn on th e day th a t th e m ateria l w as b eing ensiled. A m ong these w ere tassels, silk, som e of the upper green leaves, and som e of the low er dry leaves. E ach of these m ateria ls w as placed im m ediately in a d ry ­ ing room a t 50°—60° C. a fte r collection, and a fte r about one w eek the dried m ateria l w as g ro u n d and sto red in stoppered bo ttles a t room te m ­ p e ra tu re s u n til needed for assaying. T he vitam in D d eterm in a tio n s w ere m ade by the biological assay m ethod. T he fo llow ing w as used as the basal rachitogenic d iet: Y ellow corn m eal O at m eal ............... W h e at g lu ten Calcium ca rb o n ate Sodium C hloride . D ried y e a s t ........... 38.0 37.5 20.0 3 .0 1.0 0.5 per per per per per per cent cent cent cent cent cent T h e cu rativ e technique w as used hi accordance w ith stan d ard linete s t p rocedure and vitam in potencies w ere determ ined in term s of U. S. P. ra t u n its of vitam in D. T he m aterials to be assayed w ere in co rp o rated a t various levels in 40 gm . of the basal rachitogenic diet and fed to stan d ard rachitic rats. The re su lts of all assays w ere com ­ pared w ith th o se from ra ts w hich received a know n am ount of vitam in D in th e form of in te rn a tio n a l reference cod liver oil. (1) B e c h te l, H . E r n e s t ; H u f f m a n , C. F . ; D u n c a n , C. W . ; a n d H o p p e r t , C. A. V i t a m i n D S tu d ie s in C attle. IV. C orn Silage as a S o u rc e of V i t a m i n D for D a ir y C a ttle. J o u r n a l of D a i r y Science, in P re ss . R e p rin t fro m M ic h ig a n A g ric u ltu ra l E x p e rim e n t Statio n Q u a rte rly F e b r u a r y , 1936. B u l l e t i n , V o l . IS. N o . J, R esu lts In Table 1 are given the an tirach itic values of the various p a rts of the corn p lan t assayed. Table I. Portion of the Corn P lant A ssayed V itam in D U nits per Pound of D ry M atter* 1226 2449 2449 ♦Air dry basis. +*Ten gram s had no antirachitic action w hen fed to a standard rachitic rat. Discussion and Summary B iological assays of the corn plant collected a t th e tim e of ensiling d em o n strated th a t those p arts of the plant w hich w ere su n -d ried in the field w ere p o ten t sources of vitam in D. Of p ractical im p o rtan ce w ere th e dry leaves on the low er portions of th e plant. It w as found th a t 0.5 gm . of the dry leaves produced a n arro w continuous line of calcification across the m etaphyses of the radius and ulna in a stan d ard rach itic ra t. H ow ever, as m uch as 10 gm . o f'd rie d m ateria l prep ared from th e g reen leaves possessed no dem onstrable q u an tity of v itam in D. T hese findings are in ag reem en t w ith o th e r w o rk in th e lite ra tu re which show s th a t fresh, g reen plant m aterials are o rd in arily devoid of an tira ch itic activity, w hereas plant m aterials allow ed to dry in direct sunshine develop an tira ch itic qualities. T he d ata presen ted in T able I. indicate th a t corn silage derives a n ti­ rachitic qualities from those p a rts of the corn plant w hich w e re sundried in th e field p rio r to ensiling. Conclusion T he an tirach itic activ ity of corn silage is derived chiefly from those p o rtion s of the corn plant which w ere sun-dried in th e field p rio r to ensiling. R ep rin ted from J o u r n a l of D a ir y S c i e n c e , Ju n e, 1936, V ol. X I X , N o . 6 V IT A M IN D S T U D IE S IN CATTLE IV . C o r n S i l a g e a s a S o u r c e o f V it a m in D f o r D a ir y C a t t l e * H. ERNEST BECHTEL,** C. E. HUFFM AN, C. W. DUCAN a n d C. A. HOPPERT Sections of D airy Husbandry and of Chemistry, Michigan Agricultural Experiment Station, E ast Lansing I n previous reports from this Station, experim ental d ata have been sub­ m itted upon the an tirachitic value of sun-cured hay for d airy cattle (1), the vitam in D sparing action of certain magnesium compounds when only small am ounts of this vitam in were present in the dairy ration (2), and the essential n atu re of ra d ia n t energy in the dietary regime of the dairy calf (3). The present report is concerned with the antirachitic value of corn silage fo r d airy cattle. Hess and W einstock (4) reported th a t green plants grown in the dark contained no vitam in D but became antirachitic afte r ultraviolet irra d ia­ tion. Bethke, K en n ard and K ik (5) failed to prevent leg weakness in chicks when green, fresh red-clover was fed as 50 per cent of the ration. Steenbock and associates (6) found th a t clover hay cured in the dark was inactive antirachitically. M ellanby and K illick (7) reported th a t summergrown grass contained more of the calcifying factor th an cabbage. G-reen spinach grown in m idsumm er has been reported by Chick and Roscoe (8) to have a slight b u t appreciable antirachitic value. W hile an investigation regarding the actual feeding of fresh, green plants as a source of vitam in D to dairy cattle is lacking, the indications are th a t fresh, green pasture grasses, in general, are poor sources of antirachitic substances. As previously indicated (1), the antirachitic potency of hays is related to th eir exposure to solar ultraviolet rays. I t is the usual practice to harvest corn fo r silage when m any of the ears have become dented, the bottom three or fo u r leaves and a portion of the husk have become d ry while the rem ain­ der of the p lan t is still green. Corn at this stage of m atu rity is m ostly green plant substance and for this reason has been considered a poor source of vitam in D, although this point has not been specifically investigated. Because of the presence of sun-dried leaves, however, and other m aterial * Published with the permission of the Director of the Experiment Station as Journal Article No. 250 (n.s.). ** This manuscript is part of a dissertation presented to the Faculty of the Graduate School of Michigan State College in partial fulfillment o f the requirements for the degree of Doctor of Philosophy. Received for publication January 27, 1936. 359 360 BECHTEL* H U F FM A N ; DUNCA N AND HOPPERT on the p lan t a t the tim e of ensiling, it was thought possible th a t corn silage m ight have an appreciable antirachitic value. The object of this investigation was to determ ine the an tira ch itic value of corn silage for dairy cows. E X P E R IM E N T A L The corn silage was made from dent corn which was cu t w hen the kernels were dented and the lower leaves were dry. One sam ple of silage was taken fo r each of the following years, 1931-1934, inclusive, fo r biolog­ ical assay w ith rats. Each sample of silage was im m ediately p u t in a d ry in g room a t 50°-60° C. afte r collection and afte r about one week, th e dried m aterial was ground and stored in stoppered bottles at room tem p eratu res u n til needed fo r assaying. The vitam in D content of the silage was d eter­ m ined by the curative feeding technique w ith ra ts according to a sta n d a rd line-test procedure. The vitam in D contents of the silage, in term s of U. S. P. ra t units, are shown in Table 1. TABLE 1 Vitamin D content o f silage, U. S. P. rat im its U N IT S PER P O U N D DRY M ATTER* C U R A T IV E M A T E R IA L Corn Corn Corn Corn silage silage silage silage 1931 1932 1933 1931 ................................................ ................................................ ................................................ ................................................ 165 122122 165 * Air dried basis. The nineteen grade-H olstein dairy calves of either sex w hich were used in this experim ent were divided into 5 groups. The calves in the first 4 groups were placed on experim ent at b irth b u t the calves in group 5 were several m onths of age when placed on this experim ent. The m anagem ent of the calves and the composition of the rachitogenic calf ratio n were sim ilar to th a t previously reported (1 -3 ). Blood samples were obtained from each of the experim ental calves every two weeks and the plasm a from each sam ple was analyzed fo r calcium, inorganic phosphorus (9) and m agnesium (10) by methods already recorded. A t the tim e of post-m ortem exam ination, certain bones were saved from each anim al and studied histologically (11). The chemical analysis of the various feeds used in this investigation are given in Table 2. The calves in Group I, C-140, C-161 and C-164, were fed th e u n su p p le ­ m ented basal rachitogenic ration. The calves in G roup II, C-135, C-139 and C-141, each received the ash from one pound of d ry silage p e r day in addition to the basal ration. The calves in Group I I I , C-132, C-136, C-137, 361 VITAMIN D STUDIES IN CATTLE TABLE 2 Chemical analysis of feeds and water M A T E R IA L per cent Whole milk ...................... Skim milk ........................... Corn and oats .................. Grain mixture .................. Corn silage 1931-32* ..... Corn silage 1932-33 ........ Corn silage 1933-34 ........ Corn silage 1934—35 ........ Corn silage ash ................ Water ................................. Ca M O IS T U R E 11.80 11.30 76.61 78.02 70.51 69.00 per cent 0.120 0.122 0.128 0.494 0.175 0.101 0.103 0.126 7.190 0.0082 P Mg per cent 0.093 0.096 0.321 0.364 0.062 0.054 0.054 0.064 2.950 per cent 0.012 0.012 0.156 0.199 0.122 0.067 0.088 0.115 6.770 0.0027 ! * Each year’s analysis began with September. C-138 and C-145, received from one to six pounds of silage per day per animal. Following the above curative feeding trials, the calves in Group IV, C-156, C-159 and C-160, were used in a preventive trial. The calves in this group received the basal ration but corn silage was added as the sole source of vitam in D when the calves were 30 days of age. The calves in the above groups which survived were slaughtered at approxim ately 190 days of age, w ith the exception of C—159 which was changed to another experim ent. Calves C-168, C-169, C-188, C-195 and C-167 were subsequently added to this experim ent as Group V. . The first four calves were rachitic a t the time when silage was added to th eir basal rachitogenic ration. Calf C-168 was 269 days of age and had been suffering from rickets for about 50 days, calf C-169 was 348 days of age and had been suffering from rickets for about 100 days, calf C-188 was 428 days of age and had had rickets for more th an 200 days and calf C-195 was 395 days of age and had had rickets for at least 60 days when silage was added to their rations. Calves C-168 and C-169 were subsequently slaughtered, C-188 died while on experim ent and C-195 was continued on experim ent to determ ine the effect of the ration upon grow th and reproduction. Calf C—167 was norm al when silage was added to the ration. R E SU L T S The results obtained from the experim ent are presented in Tables 3-7, inclusive. Table 3 summarizes the data pertaining to the age when the calves were placed on experim ent, the age when the supplem ent was added, the first evidence of rickets, the age of the calves at the term ination of the experim ent and the term inal plasm a calcium, magnesium and inorganic phosphorus values. Table 4 gives the data for a representative animal in Groups I-IV , inclusive. 362 BECHTEL, H U F F M A N , D U N C A N AND HOPPERT TABLE 3 Summary of data pertaining to calves which received the rachitogenic ration Calf no. C-140 C-161 C-164 EXPER. STA RTED, AGE R A C H IT IC . AGE SUPPLEM ENT ADD ED . AGE days days 73 127 108 Group II. 0135 0139 0141 0132 0136 0137 0138 0145 1 1 1 1 1 1 1 1 Group IV. 0156 0159 0160 1 1 1 192 269 348 428 366 88 D 1 162 D 158 D 9.4 9.3 8.8 | Mg p mg. per 100 cc. 13.80 9.06 6.69 2.33 2.19 1.87 6.51 8.62 5.84 2.80 1.68 2.43 Basal ration plus silage ash 93 90 79 72 71 73 Group III. Basal ration plus silage 93 95 92 86 92 93 81 67 86 92 108 D 114 D 193 S2 193 191 192 193 190 S S S S S 6.8 7.3 8.4 8.2 8.6 8.6 10.0 10.0 3.81 3.68 3.50 7.35 2.27 2.00 2.45 2.82 2.45 Basal ration plus silage before the onset of rickets 32 33 30 Group V. 0167 0168 0169 0188 0195 T E R M IN A L P L A SM A Ca days days Group I. Basal ration none >i }> 1 1 1 EXPER. ENDED. AGE 192 269 348 428 366 161 116 95 186 S 190s 193 S 7.5 3.63 1.56 8.0 3.01 1.80 _ Basal ration plus silage (older calves) none 210 248 225 300 11404 371 S 414 S 520 S 813“ 11.9 12.3 8.5 7.27 6.79 4.13 1D denotes died. 2 S denotes slaughtered. 3 Changed to another experiment. on experiment. * Changed to another experiment, cow still alive. 3.12 2.45 2.33 * Still Group I .— The three calves in this group received the unsupplem ented basal ration. A norexia was m anifested by C-140 and C-161 a fte r th e onset of rickets and C-161 also had a convulsion a t 162 days of age. C -164 h a d a severe convulsion at 128 days of age at which tim e 5 cc. of parathorm one was injected subcutaneously. A m ild convulsion was again noted a t 157 days of age and the calf was found dead on the follow ing day. T he condi­ tion of the pen indicated th a t the calf had died in a convulsion. Group I I .— The three calves in this group h ad th e ir basal rachitogenic ration supplem ented w ith silage ash. C-135 contracted b ilatera l pneum onia 363 VITAMIN D STUDIES IN CATTLE H 03 3 bl r$ 02i p? H C3 CO tO O CO 0 3 Q M H 0 0 rH 0 3 0 3 0 3 0 3 rH 0 3 0 3 rH rH rH 0 3 0 3 03 03 03 03 0 3 0 3 0 3 C3 H H H .-2 na a> o5 CO 1C 1C 0 3 CO* CO* 1C 0 3 b - 0 0 O i H j CO 0 3 b-*t > 1C t > S rP C O fllO O O H H i CO 0 0 CO 0 0 rH 0 0 1C CO CO 1C 1C i d .§ P CD CD 1C b ’ 1C CD IQ rP tuo« 9 9 d O Growth data, mineral and silage intalces and blood plasma values of representative calves 5q | * rH CO © CO 0 3 O l> W 00 C> 0 0 0 0 io co h; i> 0 3 t JJ rH 0 5 0 0 H J 0 5 0 0 b 4 CD b - 0 0 rH 0 5 b ^ 0 5 1C O CD 03 rH rH 05* 0 0 0 0 CD 0 5 © rH © Q l> W r j N O 05 00 r j O H 0 3 CO CO t H 1C c q H j rH c o c o 0 3 0 0 0 5 03 O 03 05 05 1C O rH CO rH b - 0 5 c d b 4 0 5 o cd t 4 t4 oi o t4 i> d o dj O Tfl lO r.g r—I

* CO 0 0 0 0 t> c* 0 0 05 03 00 CD 1C © H 00 O 0 3 CO rH CD t4 05 <© CO o o 0 3 rH O b - CO CO 0 0 CO 0 5 rH rH grd O 00 . 3 3 .tT3 6 O o s's rH q-l „ O 05 rr, ’coS a c3 m ^ t-’ to d > Pn OS « P ® O p " P |§5 g I S CD Cr- H j 0 0 03* 1C 1C H* CO H j 0 5 H j O rH rH rH 0 3 CO in co h O ri H 0 3 0 3 H j CD b - A be d £g =h O 9 *03 . f^s* O CO CD 0 5 0 5 rH rH H rH H 00 05 N O 0 0 CO b - 0 0 rH 0 3 0 3 0 3 0 3 CO 0 3 03 00 05 O 0 3 1C 0 0 O 0 3 0 3 H H rH rH 0 3 0 3 0 3 o o o o ©o o CO ^D 05 03 1C oo rH H H t—i 03 Pt P P P •I O i n ~ ?„p tip c3 ^I ■a« 364 BECHTEL, H U F F M A N , D U N C A N AND HOPPERT and died at 108 days of age. C—139 had a convulsion a t 88 days of age and died at 114 days of age. C—141 had convulsions at 129 and 167 days of age. P arathorm one was injected afte r each convulsion. Convulsions were again noted at 183 and 184 days of age. ’ The calf was slaughtered a t 193 days of age. Group I I I — A fter 90 days of age, the five calves in this group received the basal ration supplem ented w ith small am ounts of silage. A ll of the calves showed the clinical evidences of rickets from 62 to 93 days of age. C-137 had convulsions at 83 and at 90 days of age. The corn silage supple­ m ent was started at 92 days of age and convulsions were observed at 128 days of age. The calves in this group were slaughtered at approxim ately 190 days of age. C-145 was representative of this group, therefore, the calcium, phosphorus and magnesium intakes and the concentration of these constituents in the blood plasm a are presented in Table 4. Group I V .— The three calves in this group had th e ir ratio n supplem ented w ith corn silage at about 30 days of age and before the onset of rickets. The calves did not develop rickets un til they were from 95 to 161 days of age. They did not consume enough silage a t an earlier age to prevent rickets b u t the silage consum ption tended to delay the sym ptoms. As they became older an insufficient am ount of silage was consumed to prevent rickets because of anorexia. C—156 and C—160 were slaughtered a t about 190 days of age and C-159 was changed to another experim ent a t 210 days of age. The representative experim ental d a ta secured from C-159 are presented in Table 4. Group V .—W ith the exception of C-167, the older calves w hich were used in this group to determ ine the antirachitic effect produced by the ingestion of large am ounts of silage were rachitic when placed on experim ent. C alf C-167. This calf had previously been used to determ ine th e efficacy of solar ultraviolet radiation in preventing the m anifestations of rickets (3). Table 5 presents the growth data, feed consum ption and blood plasm a values from 210 days of age un til the term ination of the experim ent. She received corn silage as the sole source of vitam in D at 192 days of age an d ate it w ith avidity from the very beginning, grew norm ally and m aintained a sleek appearance at all times. C-167 was first bred a t 467 days of age and again at 487 and 515 days of age. She aborted a t 601 days of age an d was again bred at 662 and 765 days of age. B eginning at 920 days of age, all grain was w ithheld from the ration and corn silage was the sole source of food other than wood shavings, salt and w ater. This heifer gave b irth to a norm al 85-pound heifer calf at 1032 days of age and had a retain ed placenta. C-167 a\erag ed approxim ately 45 pounds of milk p er day, containing 3.5 p er cent fat, during the first 100 d aj s of lactation on a ratio n w hich consisted of the rachitogenic grain m ixture, corn silage, wood shavings, salt and w ater. Pre- 365 VITAMIN D STUDIES IN CATTLE M g l> K )C |» O I> ffi« 3 H t> « « O i(O O O H N Q O t-C iS ® W O © T tl0 1 H M lO N !O K l r ) ( ( D H T j j T ) l < # q c i O H ® q q r t H » t O ( 1 0 l > t - I O M ' # O N H ’# N t D q i O b W cd ccico ca c a cd co cd cd cd cd co c d e d o a e a ca c d c a ca ca c d c d o d ed cd ca c d c d c d e d c d <3 ci W j t- fiO S ^ ft o M O H (D in m o -ii(K 3 a M (D t0 c fil0 1 .0 O C i)Q C ffiT l< 1 0 C 0 lN !a m N (N U )0 )C 0 rt N 50 M M l > 00 t o N M OS OS rH r t ® O OO O ^ O ® ffi ( p OO H W O N ^ © rH 03 oo t ~ t> t - ri-‘ cd b-‘ t- ’ t-* c o rH p o s p p p p p p p p rH p oo p p rH p p p p p ’h m h h h d r i d H H H W M n H H d d H i - i H H d d d d d H H r ir ir ir H H r H H T H H H H H H H r lH H r lH H r liH T H H r H H p p p p ffl'd r id H H H i o M g . p oo ri- p p p p as p vs a t - q r i H n q q oo r i q cij o « q ® r i « H q q © CD ri-* OS rH* O rH* rH* i-i cd rH lri Iri tri rH* Ca’ ca* rH i d i d CO* CO* CO rH* rH* rH* CO rH i-i cd 00 cd CO r H r H r H r H r H r H r H r H r H r H r H r H T H r H r H r H r H r H r H C a c a c a c a c a C a c a c O ’^ r H § ft S w 2 H a < D ,M . £ per kilo Q » . +J £'3 t> 3 • OS rH p O p p ri; p rH rH p rH O rH ri; ri; rH p ri. q s q q W N r i f f l r i O O M P rH p o r H c o * o s c a o ' c d o d o d s d o r H r H * c d c a r H c a c d o 6 i r i s d c d r H ' t r i O o ‘ c d c o " r H ' r H c d i r i c o ‘ cd 5Br H r H r H c a c a c a c a c a c a c o c o c c > c o c a c a e a c a c a c a c a c a c a c a c a c a c a c a c a c o c o c ~ i> ftJ a sio io ca o r H o sa sa sa sc o co co c o o rH k o eo o o irirH r H a sa st-o st-triO c o io co ^ rca i-H r^ o rH ca co co co co ca ca ca o o o o o ca o o o o eo ca ca co o rH o o m rH a so ^ H C O r tH n 'H lO O lO IO lO S r ir ilO C O lO r ir ia O H O H C O M H Ir iW lfiK N in c o r i H H rH rH H H H H H rlrH H rH • p rH P p rH p p 00 p p p P P p ri; P p 1- j rH rH p p rH p ri; rH p p 00 p p r j E cd tria sa siriiriiricd cd a siriiricd rH id rH 'cd cd co ’ o o o d o d c so sa s'r H o o 'a srH c a 'c a Cs H H H H H H s M £ .r H C O C O r H C D C O r H r H r H r H p p p r H p r H p p p p p p p p p p p p p p p p rH Ca 00* rH 00* rH* rH* rH* rH* SO* SO i d rH rH* rH i d i d CO CS 00 o s o o ’ ca rH ca* cd o ' ca’ CO rh r l i—I t—1 r l i—1 r l r l i—1 rH tH Silage j ft O ri; p ri; p p p O O O O p O rH p p p O p ri; p p O O ri; O p p p ri; p O ~ c d i-i c d od co* a s o ' o ' o ' o ' o ' o ' o ' rH* co* rH* as* o* cd o o ca* i d i d oo i d o ’ rH* ca* o ’ rH* i d T H rH rH rirH c a ca C a c a ca ca c a iH r H r H rlC a ca c O C O eO eO ca c O r H eH rH C O rH eH r H osoooorH rH rH rH cacacacacacacacacacacacoeocoeocoeocoeocococo r l r l l H r l H H r H H r l r l r l H i H H r H H T H r l rH tH r l r H r l r l r l r l r H r l H H ■ 5 WT. s o ooaoo o o a srH rH b -a so ca eo co rH co iriC o o so sa so rH ca co co co ca ca co co ca w •r H lO rH C O C O C D r i.lO O O C S C O r i.ca o o O t-r H cO rH O C O O C O o O C O r H r H t-r H O r H a s g rH r H O lO O O r H O r H O o o r H r H lO lrtlr tO O C a t-C O Ir iO lO O O O C O lO lO O O rH O S C a rH C O corH rH rH lO C O C O t-C -O O O O O O O O O O O O asasO O rlrlH caC acacarlO O H -H l—l i —I r l r l H r H r l H H r l H r l r l H AGE a < 0 § . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C irH rH ri-O C C C O O SC alO Q O rH rH ri-O C O C O aSC aifSaO rH rH triO O O C O aSC aiO O O rH rH 'S c a c a c a c o o ic a c c r H r H r H io io io c o c o c o c o r i.i-r io o o o o o a sa sa sa so o o r H — r lH H H -J Data from birth to 190 days of age appeared in Paper III, (3) this series. Bred at 467, 487 and 515 days of age. Aborted at 601 days of age. Bred at 662 and 765 days of age. Calved at 1032 days of age. Ca > < Vjj < . p r H O p p p p p p p r H r H r H p O r H p r H r H r H O r H p p p r H p r H p p r H r i; os’ o ' ca CO ca* rH* rH rH SO* OS rH* rH rH* 00 CO rH CO* 00* ri-’ rH* rH* CO CO* CO* 00* CO* i-i Ca* OS* rH* ri.' ri.’ H r l i—I H H H H H H N N K l i H i — H r H r l r l H H r H H H i —1 i—I r H r H i —1 rH rH rH Cs 1 A < Q 366 BECHTEL, H U F F M A N , D U N C A N AND HOPPERT lim inary results indicate th a t the m ilk produced by C—167 contained con­ siderably less th an 2.7 U. S. P. vitam in D u n its p e r q u art. The blood plasm a data fail to reveal any significant variations from norm al. H ow ever, there were two grad u al drops in the inorganic phosphorus values. The first drop was associated w ith the n a tu ra l physiological disturbance due to abortion and the second was due to p artu ritio n . C alf C -168. This calf received from 15 to 20 pounds of corn silage per day as the sole source of vitam in D from 269 days of age u n til the end of the experim ent. The calf was suffering from severe rickets w hen first placed on experim ent and the blood calcium and inorganic phosphorus values were 7.6 and 9.84 mg. per 100 cc. of plasma, respectively. The silage was readily consumed from the beginning and at 303 days of age the calf was able to rise to its feet w ith much less difficulty th an a week earlier. T he plasm a calcium and inorganic phosphorus values were 9.3 and 5.48 mg. a t 303 days of age and by the following week they had re tu rn e d to norm al, 10.9 an d 6.76 mg., respectively. The gains in body w eight were also g re ater fro m this tim e on u n til the anim al was cured of rickets and slaughtered a t 371 days of age. The term inal calcium and inorganic phosphorus values w ere 11.9 and 7.27 mg. The kidneys showed extensive areas of scar tissue. C alf C-169. This calf received 15 pounds of corn silage p e r day as the sole source of vitam in D beginning a t 348 days of age. I t was severely rachitic and had difficulty in rising to its feet. The blood calcium an d inor­ ganic phosphorus values were 8.0 and 5.84 mg. per 100 cc. of plasm a. The corn silage was readily consumed and 10 days la te r the calf was able to rise to its feet w ith much less difficulty. The blood values were approaching th eir norm al concentrations by 370 days of age and the calf began to gain in body weight. C-169 was cured from rickets when slaughtered a t 414 days of age and the term inal calcium and inorganic phosphorus values were 12.3 and 6.79 mg. The kidneys showed more extensive areas of scar tissue th an observed in C-168. The d ata are tabulated in Table 6. Calf C—188. This calf was 428 days of age and h ad been suffering from rickets fo r more th an 200 days when corn silage was added to the ra tio n as the sole source of vitam in D. The anim al was severely rach itic a t this tim e and was only m aintaining a constant body weight. The blood calcium and inorganic phosphorus values were 7.2 and 7.06 mg. p e r 100 cc. of plasm a. The silage was readily eaten b u t the grain was refused p a r t of th e time. By 472 days of age the anim al was extrem ely stiff and lam e and was ra re ly seen standing so th a t it became necessary to place the feed in a basket on the floor of the stall. The plasm a calcium had now increased to 7.8 mg. b u t the inorganic phosphorus had declined to 4.51 mg. P lacing the feed before the anim al resulted in an increase in silage consum ption b u t no im provem ent was noticed in its well-being. A t 504 days of age the anim al was observed 367 VITAM IN D STUDIES IN CATTLE co cq MJ 0 1 co eq co cq n «] o ra in c- M (O Mi Mi 10 10 to -e © A © + » O 0 2 CD .2 368 BECHTEL, H U F F M A N , DUN C A N AND HOPPERT struggling to its feet, breathing h ard and w ith nostrils distended. The following week C—188 was emaciated, unable to rise to its feet a n d refused all feed and water. The plasma calcium and inorganic phosphorus values at this time had declined to 7.1 and 2.44 mg. The animal lapsed into a coma­ tose condition at 518 days of age and failed to respond appreciably either to intravenous injections of solutions of calcium gluconate, glucose an d m ag­ nesium sulfate or to a subcutaneous injection of viosterol. D eath occurred at 520 days of age. There were patches of scar tissue in the kidneys, the muscle tissue showed considerable edema and there were evidences of muscle in ju ry around the leg joints. The most characteristic autopsy finding was the generalized condition of pitting and erosion of the artic u la r surfaces of the long bones. Illustrations and a detailed description of the histological findings are given elsewhere (11). Calf C-195. This calf was 366 days of age and had suffered from rickets for more than 60 days when corn silage was added to th e rachitogenic ration as the sole source of vitamin D. The an im al’s legs were stiff and the knees were bowed markedly. The plasma calcium and inorganic phosphorus values were 9.5 and 3.01 mg. per 100 cc. at this time. The appetite for silage was only fa ir but it gradually improved and by the following month the calf was able to rise to its feet with much less effort. The addition of silage had little effect upon the blood constituents d u rin g the first two months b ut during the next two months, the concentration of calcium returned to normal and the inorganic phosphorus manifested a slow but steady rise. E stru s was first noted at 413 days of age, at which tim e the bowing of the knees had become less marked. Im provement was ra p id after that time. C-195 had recovered from rickets by 543 days of age, was bred and thereafter made normal gains in body weight. The heifer gave birth to a normal 82-pound bull calf at 813 days of age. The d a ta secured from this animal are summarized in Table 7. DISCUSSION W hen calves less than 190 days of age were used as test animals it was impossible to either cure or prevent rickets by supplem enting the basal rachitogenic ration with corn silage. A larger percentage of the calves survived until 190 days of age, however, when silage supplem ented the basal ration (Table 3). This suggested that silage exerted some antirachitic activ­ ity although the amount ingested was insufficient to m ain tain or to restore health. H igher intakes of silage were precluded by anorexia. Calf C-145 had the best appetite for silage b ut the average consumption was less th an six pounds per day. This amount of silage was equivalent to a daily intake of approximately 5 grams of silage dry-m atter p er kilo of body weight and was ineffective in curing rickets. The ingestion of three pounds of corn silage per day failed to prevent rickets in young calves. 369 VITAM IN D STUDIES IN CATTLE wiomwooojoominffioiaooffi'nt'NNffiMot) cq pHin in to in ^ 03 co pHo in co os oo w in ao t> in co in oa en ©a' ca' ea' ca ©a ©a ©a ©a ca ©a ©a ©a' ©a ca' ©a' ©a' ©a' ca ©a ©a' £ inOH ^ H| « t - n m o hj n io t£ io toa o t- o b; oo« os inpH » id id '■#'-d co ©a ©a edid id in*id id id id id id id in' in' d> ___________________________ ________ 5 os’ oi o r i o h hh h hh h h ri o' o CO00lOhHhHin OSt- C©pHH*©3t-HOSQ OS!>; e©00©3 t-'t^t^t>t^cdco'co'ed'tf*fc^ido5c^t^odcsoiodo6 riHpHHHpHHpHpHpHHHHHrl 03 ©a 03 03 03 ID pHOS O to IN OS O IQ 00 03 c©co co’ co co id id in' id id in' tjhoo" in 03 ID b-- id id id id id O } • cq io cc iq cvi co t- i-hh .n oo cot- h ^ o w oq fj_ ca cs oo cd oo*v i iri 10 ?o w d o’ «o ts oooo n to ^ H H H H H rlH H H H H H rlH H H H H H H riHHHHWNNN«MNN(MW(MN-O^O^COC5 ^ICLOr^ldt^tCt^t^t^GOoicjOOOt^ Growth silage ■CO^O’^tOOO^HTfilQfOOiCOOqt> lOt0^lOt>OJt>ffJOiXOOOOCi t— iH H H rHlo 00 l>■1£3 00 1Ci O CI00 05 03 C5 CQ05 lO 05 W ^ O CON O 50 O H W tO r | 50 i> o d oo oi h ci d c oi d o' qi o! oo Ef H3 . CO p^ ^odt>lOOiC0O5Tf5'^l£Jfcs -G©t**C,Dl£) HHHHrtWHCSHMQQNWCo lO o o pHpHpHpHpHpHpHpHpHpHpHpHpHpH and plasma values O O Wt»_rj >o n o' o' d o a H H pH pH _ g H H rlH H 00OH(MinWW^l>?0

t> m dns) rlH H H riH H rlH H H H H COHr ctf ih ? to in bn® += c^'H ° o m cS £*HTHb-?OWO^NOlOOUJ^OCONcOHH t-pH03M'<*lHH-HSC0l0t-03int-OC3l>-iM'^t»pH0a T tiininininm ininininioiD iot-t-t-ooooooosos iS M rp ^He© ° § 10 "cs +1 “ S Im n ro ■M 4-3 M 3 I© > S? o o o o o m o o o o o o o o o o o o o o o c o T tlt^O C 0l0l0C S03in00rH T flt~ O C 0lD 05C 3in00rH pH Sg > o]03cncocococo-#T^H|iinininiocDiDiDt-t-t'-ooQo Cj +3 © ,—| •S » JS ei 02H M o 370 BECHTEL, H U F F M A N , DUN C A N AND HOPPERT W hen older calves were used, the clinical manifestations of rickets were alleviated in three out of four eases by the daily ingestion of 15 to 20 pounds of silage (Tables 6—7). Backets was also prevented in C—167 from six months to three years of age by the ingestion of corn silage (Table 5). The abortion of C-167 was probably not associated w ith a vitam in D deficiency in view of the results reported by Moore and associates (12). The decrease in the concentration of inorganic phosphorus in the plasm a of C—167 and C-195 at the time of parturitio n and the abortion of C-167 was associated with the physiologic disturbances caused by these acts ra th e r th a n by a deficiency of the antirachitic factor. I t has been shown (9) t h a t such a change in the composition of the blood plasma is not uncommon in dairy cattle at the time of parturition. I n order to compare the results obtained with the various animals it seems convenient to express the intake of corn silage in term s of gram s of silage dry-matter per kilo of body weight. On this basis, 7 to 10 gm. of silage dry-matter per day was not only effective in curing and preventing rickets but also supplied a sufficient amount of the antirachitic factor for normal growth and reproduction. Calf C-188 ingested from 15 to 20 pounds of silage p er d ay which was equivalent to 8 to 12 gm. of silage dry-m atter per kilo of body weight, yet failed to recover from rickets. This was the only one of the older calves which failed to respond favorably to silage feeding. This calf is regarded as an atypical case. The inability to eure C-188 is ascribed to the severity of the rachitic condition which had developed before curative measures were begun. F ailure to utilize its feed properly, together with its emaciated appearance and autopsy findings in the joints, indicate th a t the animal was suffering from far-advanced rickets which had given rise to other n u tr i­ tional disturbances refractory to ordinary vitam in D therapy. H u ty ra and Marek (13) state th at if rickets is fa r advanced and has given rise to emphatic nutritional disturbances, death always follows, either through exhaustion or through some complication. The ultim ate healing of the process is frequently prevented by ulcers formed in the course of the disease in the articular cartilages. Bioassays made with rats indicated th a t 7.5 to 10.0 gm. of d ry corn silage contained an equivalent of 2.7 U. S. P. units of vitam in D (Table 1). Assuming th a t 10.0 gm. of dry silage contained 2.7 U. S, P. r a t units, d uring the period 1931-1934 inclusive, the daily intake of vitam in D un its was calculated for each of the experimental calves (Tables F-7). The representative tables included in this pap e r show t h a t the addition of corn silage to the rachitogenic ration appreciably increased th e intake of magnesium. In view of the results of a recent investigation (2), it is possible th at the presence of magnesium in silage m ay augm ent the efficacy of the antirachitic material in corn silage. VITAMIN D STUDIES IN CATTLE 371 SUM M ARY AND CONCLUSIONS 1. I t was the purpose of this investigation to determine the antirachitic value of corn silage for dairy calves by the use of both curative and p re ­ ventive feeding trials. 2. Samples of corn silage for the years 1931-1934 contained from 122 to 165 U. S. P. vitamin D units per pound of dry matter. 3. Calves less than 190 days of age were unsuitable test animals for both curative and preventive trials because of anorexia and failure to ingest adequate amounts of silage. In one instance, the feeding of corn silage to a yearling calf failed to cure severe rickets complicated with muscle atrophy and erosion of the articular surfaces. 4. The daily ingestion of an equivalent of 7.0 to 10.0 gm. of dry corn silage per kilo of body weight was effective in curing and preventing rickets in yearling calves and also supplied sufficient antirachitic material for normal growth and reproduction in dairy cows. 5. When corn is cut at the usual stage of m aturity for corn silage it possesses definite antirachitic qualities when fed to dairy cows. The writers are indebted to Mr. C. C. Lightfoot for technical assistance in the determination of the blood values and to Mr. 0. B. W inter and Miss Lillian I B utler for the chemical analyses of the feeds. LITERATURE CITED 1. H o f f m a n , C. F., a n d D u n c a n , C. W. Vitamin D studies in cattle. I. The anti­ rachitic value of hay in the ration of dairy cattle. J o u r n a l of D a ir y S c ie n c e 18: 511. 1935. 2. , A N D ------------------- . Vitamin D studies in cattle. II. The vitamin D s p a r in g a c tio n o f m a g n e s iu m in th e r a t io n o f d a ir y c a t t le . 3. 4. 5. 6. 7. 8. J o u r n a l of D a ir y 18: 605. 1935. D u n c a n , C. W., a n d H u f f m a n , C. F. Vitamin D studies in cattle. III. Influence of solar ultraviolet radiation upon the blood chemistry and mineralmetabolism o f dairy calves. J o u r n a l of D a ir y S c ie n c e , 19: 291, 1936. H e s s , A. F ., a n d W e i n s t o c k , M. Antirachitic properties imparted to inert fluids and to green vegetables by ultraviolet radiation. J. Biol. Chem. 62: 301. 1924. B e t h k e , R. M., K e n n a r d , D. C., a n d K i k , M. C. Nutritional studies of the growing chick. I. Relation of sunlight and green clover to leg weakness in chicks. J. Biol. Chem. 63: 377. 1925. S t e e n b o c k , H., H a r t , E. B., E l v e h j e m , C. A., a n d K l e t z i e n , S. W. F. D ie t a r y factors influencing calcium assimilation. VI. The antirachitic properties of hay as related to climatic conditions with some observations on the effect of irradiation with ultraviolet light. J. Biol. Chem. 66: 425. 1925. M ellAnby, M., and K illicic, E. M. CXIII. A preliminary study of factors influenc­ ing calcification processes in the rabbit. Biochem. J. 20: 902. 1926. C h ic k , H., a n d R o s c o e , M. H . The antirachitic value of fresh spinach. Biochem. J. 20: 1. 1926. S c ie n c e 372 BECHTEL, H U F F M A N , D U N C A N AND HOPPERT 9. H u f f m a n , C. F ., D u n c a n , C. W., R o b i n s o n , C. 8 ., a n d L a m b , L . W. Pliosphorus re­ quirement of dairy cattle when alfalfa furnishes the principal source o f protein. Mich. Agr. Exp. Sta. Tech. Bull. 184. 1933. 10. D u n c a n , C. W., H u f f m a n , C. F., a n d R o b i n s o n , C. S. Magnesium studies in calves. I. Tetany produced by a ration of milk or milk with various supplements. J. Biol. Chem, 108; 35. 1935. 11. B e c h t e l , H . E., H a l l m a n , E. T., H u f f m a n , C. F., a n d D u n c a n , C. W, Pathology of rickets in dairy calves. Mich. Agr. Exp. Sta. Tech. Bull. 150. 1936. 12. M oore , L. A., H u f f m a n , C. F., a n d D u n c a n , C. W. Blindness in cattle associated with a constriction of the optic nerve and probably of nutritional origin. J. Nutrition 9: 533. 1935. 13. H u t r y a , F., a n d M a r e k , J. Special Pathology and Therapeutics of the Diseases of Domestic Animals. Alexander Eger, Chicago 3: 244. 1926. T echnical B ulletin No. 150 May, 1936 PATHOLOGY OF RICKETS IN DAIRY CALVES 7 H. E R N E S T B E C H T E L , E. T. H A L LM A N , C. F. H U F FM A N '' A N D C. W . D U N C A N AGRICULTURAL EXPERIMENT STATION MICHIGAN STATE COLLEGE O f Agriculture and A pplied Science SE C T IO N S O F D A IR Y H U S B A N D R Y , A N IM A L PATH O LO G Y, A N D C H E M IST R Y E ast Lansing, Michigan T e c h n i c a l B u l l e t i n N o . 150 M a y , 1936 PATHOLOGY OF RICKETS IN DAIRY CALVES H. ERNEST BECHTEL, E. T. HALLMAN, C. F. HUFFMAN AND C. W. DUNCAN AGRICULTURAL EXPERIMENT STATION MICHIGAN STATE COLLEGE Of Agriculture and Applied Science SECTIONS OF DAIRY HUSBANDRY, ANIMAL PATHOLOGY, AND CHEMISTRY E a s t L a n s in g , M ic h ig a n TABLE OF CONTENTS Page n trod u ctio n ......................................................................................................... -3 Materials and M e t h o d s ............................ ...................................................... ^ lesu lts .................................................................................................................. C haracteristic S y m p to m s of V itam in D Deficiency in C a lv e s . . ^ 5 T h e N orm al C ostochondral J u n c t i o n ................................................ 5 1. Zone of R eserv e Cells *• • • 5 2. Zone of Cell P ro life ra tio n .............................■'................... 6 3. Zone of Cell G ro w th ............................................................... 7 4. Zone of M a tu re C e l l s ............................................................. 7 5. Zone of C artilage R em o val ............................................... * 6. Zone of Ossification ............................................................... 7. Zone of Com pact S ub stance .............................................. T h e Costochondral J u n c tio n in R i c k e t s .......................................... 1. Zone of R e s tin g C artilage .................................................. 2. Zone of Cell P ro life ra tio n ................................................... 3. Zone of Cell G ro w th ............................................................. 4. Zone of M a tu re Cells ............................................................ 5. Zone of Cartilage R em o v al ................................................. 6. Zone of O s s i f i c a t i o n ................................................................ 8 8 10 10 H 11 11 11 12 14 7. Zone of Com pact S u bstance ............................................... T he C ostochondral J u n c tio n in H e a lin g R ic k e ts ...................... R o e n tg e n o g ra p h ic A ppearan ce of N o rm a l and R ick etic R ib s. G ro w th as a M odifying F a c to r ......................................................... C om plications in R ick ets ...................................................................... T y p e of R ickets and Plistological A lte ra tio n s ............................. D i s c u s s i o n ............................................................................................................. S u m m a r y ......................................................................................... ; ................... Conclusions ........................................................................................................... L ite ra tu re Cited ................................................................................................ 14 15 15 16 16 17 17 19 21 22 Tables .................................................................................................................... Illustrations ......................................................................................................... 23 32 P A T H O L O G Y O F R IC K E T S IN DAIRY CALVES H . E R N E S T B E C H T E L , E . T. H A L L M A N , C. F. H U F F M A N A N D C. \ V . D U N C A N INTRODUCTION Recognition of the im portance of vitamin D in dairy calf nutrition has resulted in considerable w o rk on the production and prevention of rickets in calves by the Michigan (1-6), the Pennsylvania (7) and the W isconsin A gricultural E xperim ent Stations (8). This report p ri­ marily- concerns results of a study of the pathology of ricketic bones produced in calves at the Michigan A gricultural E xperim ent Station. F ew histological data are available which deal with rickets in calves. H u ty ra and M arek (9) presented a good clinical picture of calf rickets b u t reported nothing concerning- the histological findings, although th e y included several general anatomical changes which w ere due to rickets. These investigators also noted th a t the bone development of a one-year-old child corresponded to the bone development of a calf during- fetal life. F ro m this observation, they concluded th at calf rickets and late rickets in children were similar. M arek and W ell­ m an (10) reported considerable d a ta 'u p o n the histology of rickets in sheep and swune but relatively few data upon calves. The Pennsylvania station (7) presented a brief description of a photom icrograph of the n inth costochondral junction taken from a ricketic calf. No histological s tu d y of calf rickets was included in the report from the Wisconsin station (8). T heiler’s (11) w ork in South Africa was concerned with aphosphorosis in heifers betw een two and three years of age, so th a t his photom icrographs are not directly applicable to the present study because of the age of his animals. H arris (12) has published extensively on hum an rickets. Maxwell, H u and Turnbull (13) have described in detail the histology of a case of fetal rickets in the hum an being. The histology of rickets in the ra t has been studied by Pappenheim er (14) and Dodds and Cameron (15) ’who have stated th a t the histological pictures in hum an and in ra t rickets are fundamentally identical. The w o rk of the latte r group of investigators has been a helpful guide in th e present histological study. P au city of fundam ental data in the literatu re concerning the histol­ og y of rickets in dairy calves is the justification for reporting the pres­ e n t study in detail. M ICH IGA N T E C H N IC A L B U L L E T IN NO. 150 MATERIALS AND METHODS M o r e t h a n 1 0 0 d a i r y calves h a v e b e e n u s e d in t h e e x p e r i m e n t s o n low' v i t a m i n D r i c k e t s w h i c h h a v e m a d e r i c k e t i c b on e s a v ai la bl e f o r this Avork. F r o m t hi s g r o u p , five n o r m a l a n d e le ve n r i c k e t i c ca lv e s w e r e s e le ct ed f or t h e d et ai le d h i s t ol o gi c a l s t u d y w h i c h is p r e s e n t e d in t hi s bulletin. T h e m a j o r i t y of t h e s ele ct e d case s Avere m a l e calves o f g r a d e H o l s t e i n b r ee di ng - a n d v a r i e d f r o m 1 5 1 t o 5 2 0 d a y s of a g e a t d e a t h . T h e d u r a t i o n of r ic ke ts, as in di ca te d b y t h e b lood p l a s m a a n a l y s e s f o r c alc iu m a n d i n o r g a n i c p h o s p h o r u s , v a r i e d f r o m 3 8 t o 2 3 9 d a y s so t h a t thi s g r o u p r e p r e s e n t s m a n y s t a g e s in t h e s e v e r i t y of t h e disease. E a rly in the investigation it becam e a p p a re n t t h a t th e co sto c h o n ­ dral junction at the v en tra l end of th e rib wras th e best index to th e severity of rick ets because it showed to a g r e a t e r deg ree the sam e changes displayed by the hum erus, femur, m etac arp u s and m e ta ta r s u s . M id-frontal sections of about 4 mm. in thickness w e re ta k e n from the last th ree inches of the v e n tra l end of the left eighth rib of each calf. These sections w ere studied by the com parison of r o e n tg e n o ­ gram s, p h o to g rap h s of specimens stained in silver n it r a t e solution, and histological sections. A f t e r c o n s id e r a b le a d o p te d in th e s p e c im e n s ru n n in g w a s h e d T h is w e r e fix ed w a t e r , a a q u e o u s few' h o u r s p r o c e d u r e c a tio n , o t h e r tio n o f AA'hich A v a s Avas o s te o id u s e d s e c tio n s a lso s ta in e d t h ic k n e s s a c id u la te d a p e r o f a n d p e r u s u a l a n d to lie less e ig h t p e r a lc o h o l c e n t a n d m u c h Avere flat a n d s o lu tio n , e o sin a c e tic in s ta in e d a cid cc. th e o f slides. th e T h e o f b y s o lu tio n S u c h h e m o t o x y l i n in w e r e t h r e e u p liver, t h e a t H a r r i s ’ s e c o n d s in five b y th e s e c ­ c u t in a s e o s in t e c h ­ t i m e - c o n ­ g e t t i n g c o n ta i n e d a n d s e v e r a l c e llo id in m a d e tis s u e s d e ­ d i f f e r e n t ia ­ Avas fo r f o llo w e d t h e d e c a lc ifi­ a ls o s ta in e d Avas c e n t so ft. i m b e d d i n g T h e s ta i n in g e o s in s o lu tio n , of in h o u r s , p e r w 'h e n s e c tio n s r e g r e s s iv e ly h o u r s 5 a n d i m b e d d i n g T h e w e r e s ta i n in g p a ra ffin Avas : 2 4 a m e t h o d e n c o u n t e r e d t h e so lu tio n . Z e n k e r ’s in Avas folloA ved 50 w re e k s m e t h o d T h e M o s t s o lu tio n . E a c h a c id o n Avere six T h is C e llo id in difficulty p e r f e c tly to e x c e lle n t tis s u e s. p e r m a n e n tl y . fixed C. tis s u e s Aveek. e x c lu s iv e ly . in 37° f o u r in f e w le a s t t h e f r o m n itric a a t a t e v e r y th e fo r s tu d ie s u n til r e q u ir e d o s s e o u s w 'a s h e d a lc o h o l d e c alc ified r e s u lte d t e c h n i q u e h is to lo g ic a l f o rm a lin , c e n t d i c h r o m a t e to fo llo w d n g f o r r e n e w e d so lu tio n . g lac ia l Avere p e r a n d m ic r o n s c e n t t h e s e c tio n s c e n t 80 w a t e r , a n d h e m o t o x y l in sp le e n te c h n iq u e th e o f o n e -h a lf c e n t 2 p e r in w 'o rk t h e s u p e r io r a lm o s t la b o rio u s , in w a s fa r cello id in a 10 d e c a lc ify in g - s o lu tio n s , s u m in g , tio n s in of p o t a s s i u m u s u a lly s o lu tio n betA veen n iq u e in h a r d e n e d s o lu tio n c a lc ify in g p r e l i m i n a r y p r e p a r a ti o n a 7 0 d r o p s k i d n e y p a ra ffin s o lu tio n s in m a n n e r . RESULTS The ages of the calves at death, the d u ra tio n of the rick etic condition and o th e r d ata p e rta in in g to the anim als used in th e study of bone histology are sum m arized in Table 1. T h e groAvth d a ta and th e c o n ­ c e n tratio n s of calcium, inorganic ph osp h o ru s and m a g n esiu m of the blood plasm a of each individual calf are p re s e n te d in Tables 2-9, in ­ clusive. PATHOLOGY OF RICKETS IX DAIRY CALVES Characteristic Symptoms of Vitamin D Deficiency in Calves In this investigation the decrease in the concentration of calcium a n d /o r inorganic phosphorus of the blood plasma was am ong the first signs of low vitamin D rickets. The most conspicuous post m ortem findings, however, were those which apparently accompanied the blood changes and which occurred in the bones. Clinically, the skeletal changes included bow ing of the forelegs either forw ard or to the side, swell­ ing of the knee and hock joints, straig htening of the pasterns, occa­ sional ring-like swellings on the pasterns, and hum ping of the back (Fig. 1). P o ste rio r paralysis occurred in cases of fractured vertebrae. F rac tu red femora sometimes occurred. Other symptoms frequently observed were stiffness of gait, dragg ing of the rear feet, standing with the rear legs crossed, irritability, tetany, rapid respiration, bloat, anorexia for grain and roughages but not for milk, weakness and in­ ability to stand for an)- length of time, and finally the retard ation or complete cessation of g ro w th in bod)' weight. The post m ortem examinations of the ricketic calves showed th at the principal alterations were confined to the skeleton. The liver, kid­ neys and spleen were negative, but the gall bladders frequently con­ tained as much as 500 cc. of a viscous (sometimes ropy) orange to yellow colored bile. The gall bladders of normal calves contained less than 100 cc. of am ber to olive-green colored bile. Bile stained ingesta occurred in the upper part of the small intestine in a few cases. E n teritis was also occasionally seen. Excessive accumulations of syno­ vial fluid in the joints were frequently observed in the more ricketic calves. This m aterial was especially abundant in the larger joints and varied from a thick, viscous fluid to a heavy, jelly-like substance. The alterations in the skeletons may be more readily understood by first studying the process of osteogenesis as discussed by Maximow and Bloom (16) and by Leriche and Policard (17). An understanding of the origin and importance of the epiphyseal or interm ediary cartilage is especially desirable. The Normal Costochondral Junction The following detailed discussion of the normal costochondral junc­ tion will serve as a w orkin g basis in interp retin g the histological pic­ ture of ricketic bone. This description is based on material from five norm al dairy calves which were between 161 and 317 days of age at the time of slaughter. F or convenience of description the ventral-epiphyseal end of the rib, beginning at the union of the rib with the sternum and proceeding vertebrallv, was divided into seven zones. The first five of these zones are according to the divisions suggested by Dodds and Cameron (15) for the epiphyseal cartilage (Fig. 3). 1. Z o n e o f Reserve Cells—This area comprised the last several milli­ m eters of the ventral end of the rib and usually term inated in a m oder­ ately concave fashion where it joined the second zone. The tissue was typical hyaline cartilage and was traversed in several places by vascular bundles. "the diam eters of which were sometimes as g reat as 0.75 mm. (Fig. 3). The num ber of vascular bundles was g re ater when the sec­ ond^zone was approached. There were indications, as referred to below, M IC H IG A N T E C H N IC A L B U L L E T IN NO. 150 th a t th e blood vessels in th e cartila g en o u s end of th e rib a n a s to m o s e d w ith the vascular sy stem in th e diaphysis. C artilage cell lacunae w e re irre g u la rly sca tte red , usually only p a r tly filled by th e contained cells, and becam e m ore n u m e ro u s as th e second zone w as approached. T h e y varied from oval o r cresce n t form s to m o re spherical shapes which ch a rac te rized th e m a tu r e cells (F ig. 4). L a ­ cunae adjacen t to the la rg e r vascular bundles w e re fre q u e n tly flatten e d w ith th e ir long 'axes tra n s v e rs e to th o se of th e bundles. T h e y o u n g , crescen t-sh aped cells w e re usually gro u p e d in pairs, except in th e v e n ­ tra l-p e rip h era l regio ns of Zone 1 w h e re as m an y as eig ht cells o c c u rre d in one group. W h e n a r ra n g e d in isogenous pairs— and this see m e d to be m ore freq uen t n ea r th e second zone— th e c resce n t shaped cells w e re s itu ated w ith th e ir concave surfaces directly apposed to each other. In this p a r t of Zone 1, th e in te rs titia l sub stan ce b e tw e e n is o g e n ­ ous pairs of cells w as som etim es invisible w h e n view ed in one plane, as in a section, and tw o im m a tu re cells app e are d to be in one oval-sh ap ed cavity (Fig. 4). Individual cartilag e cells, w h e n n o t lost or sh ru n k e n , c o n fo rm ed in shape to th e ir respective lacunae. T h e m a tu re r e s tin g cell, w h e n it filled its lacuna, av e rag e d a b o u t 20 m icrons in lo n g e s t d iam eter. T h e cytoplasm was reticular, m o d era tely basophilic, and con taine d several vacuoles. T h e nucleus, w hich w as basophilic and reticular, w a s a b o u t six m icrons in d iam eter and contained several c h ro m a tin g ran u le s. C artilage cells w e re occasionally found which s u g g e s te d sta g e s in m i to ­ sis, while o th e r cartilag e cells appeared to be in v ariou s s ta g e s of degeneration. 2. Zone of Cell Proliferation — P ro ce ed in g fro m Zone 1 t o w a r d th e diaphysis th e re w e re m an y pairs of isogenous cells a r r a n g e d p r e ­ dom inately a t first and la te r exclusively w ith th e ir lo n g axes p e r p e n ­ dicular to the long axis of th e bone. T his m a rk e d th e b e g in n in g of Zone 2 (Fig. 3). D a u g h te r cells, w h e n th ey o rig in ated in this area, ap p e are d to u n d e r ­ go im m ed iate division and g ave rise to fo u r cells. T his pro cess usually continued and produced 8, 16, or m o re cells in one isogenous group. F o r th e eig h th costochondral ju nction of th e d airy calf, th e n u m b e r of such cells in one g ro up w hich appeared m o s t freq u en tly , a p p ro x i­ m ated 32 cells— indicating t h a t each p a r e n t cell h ad divided five tim es. This n u m b e r was variable, how ever, and it w a s difficult to d e te r m in e ac cu rately w hen the cells w e re so closely apposed to each oth er. T h e y frequ en tly failed even to ap p ro x im a te a m ultiple of tw o. T his m ay have been caused by th e plane of sectioning, or possibly th e g ro u p w a s not fully developed a t the tim e of tissue fixation or t h a t c e rta in cells in such a g roup divided m o re actively th a n others. Cells in th ese isogenous g rou ps continu ed to a r r a n g e th em selv es d u rin g pro liferatio n w ith th e ir long axes perp en d icu lar to th e lo n g axis of th e bone and piled one above th e o th e r so t h a t th e lo n g axis of th e g ro u p w as parallel to th a t of the bone. T hus, a g ro u p or colum n of cells arose from one p a r e n t cell. T h e s e colum ns ex ten d e d ac ro ss th e bone w ith each colum n s e p a ra te d from its n e ig h b o r by h o m o g e n e o u s in terstitia l substance. A dditional colum ns aro se and a r r a n g e d in m o re or less ta n d e m fashion to form a ro w co n sistin g of g ro u p s o r colum ns of cartilage cells. As n e w g roup s aro se th e older ones w e re seem in g ly PATHOLOGY OF RICKETS IN DAIRY CALVES 7 pushed on to w a rd the diaphysis of the bone. Groups within row s w ere sep arated by a thick mass of interstitial m atrix in contrast to the thin tran sv e rse walls betw een cells within any one group. C artilage cells in this zone were approxim ately of equal size and stage of im m aturity. In th e axial p a rt of the bone this zone term in ated on its v erte b ral side in a concave m anner as it m erged into the n ex t zone. This concavity som etim es continued across the entire bone, in which case Zone 2 had a uniform depth, but in m ost cases this zone becam e deeper as it extended from the axial area tow ard th e peripheral p a r t of the bone and usually attained m axim um depth a t 1.5 to 4.0 mm. b en eath th e perichondrium. Zone 2 averaged about 600 microns in depth in the axial region of th e bone and occasionally became as deep as 1,300 m icrons in the peripheral p a r t of the rib. 3. Zone of Cell Growth—This zone was identified in two ways, first, by th e g ra d atio n in size of cells, and second, by the gradual thinning of th e interstitial m atrix (Fig. 3). G row th of the cells in this zone w as apparently limited a t first to thickening in a plane parallel w ith th e long axis of th e bone. L ater, w hen their thickness equaled their width, these cells seemed to enlarge in all directions and ceased g r o w ­ in g w hen they attain ed an av erage diam eter of more than 30 microns. G ro w th resulted in crowding and m utual pressure. T he cells as­ sum ed m ulta n g u la r shapes, while th e m atrix betw een the cells w ithin a g rou p gradually disappeared and the columnar a rran g e m e n t w ithin th e isogenous groups became less perfect. There was lateral expansion of these groups which involved a thinning of the interstitial m atrix betw e en the rows. M a tu re cells in this zone, except for their larger size and absence of im m a tu re forms, w ere similar to those in Zone 1. In fixed section, in about th e last 100 microns as Zone 4 was approached, th e cartilage cells occupied a decreasing percentage of the areas in their lacunae. The nuclei appeared shrunken, the cytoplasm increased in density, p a r ­ ticularly in the region of the endoplasm w here a condensation w a s som etim es observed, definitely outlining and surrounding the nucleus. S everal small vacuoles appeared in the cytoplasm. As these degenera­ tive processes continued, the outline of the nucleus disappeared and finally m ore than the usual num ber of em pty lacunae became visible. T hus, while g ro w th was the predom inating feature of this zone, it was nevertheless superseded in many, bu t not all, of the cells by w h a t has been called hypertrophic degeneration. 4, Zone of Mature Cells—In the norm al rib, the m ost characteristic fe atu re of this zone was calcification of the interstitial m atrix. This calcified m atrix was later removed in its entirety. F o r this reason, this is som etimes called the zone of tem p o ra ry or provisional calcifica­ tion. This zone followed the outline of the previous area and showed a continuation of the same degenerative changes which had not gone to completion even in this zone (Figs. 3, 7, and 9). In proceeding vertebrally th ro u g h this zone, coarse granules of a basophilic substance w e re observed in the interstitial matrix. As these granules increased in num ber, they produced a stippled effect. L a te r they becam e obscured as they increased in num ber and became m o re closely packed. This basophilic substance m arked the location of in­ M ICH IGA N T E C H N IC A L B U L L E T IN NO. 150 organic salts. The salts w e re deposited in all of th e lon g itu d in al walls between th e row s of cells and b etw e en th e g-roups of cells w ith in the rows, b u t never betw e en the cells w ithin th e isogenous g ro u p s because here th e in terstitia l m a trix appeared to be absent. T his point is i n t e n ­ tionally em phasized because of some debate over it in th e lite ra tu re . Calcification w as o ften m ore com plete in th e m a rg in s t h a n in th e axial areas of the longitudinal walls of ca rtila g e m a tr ix (Fig. 9). This zone av e rag e d a b o u t 100 m icrons in depth. In a b o u t half th e rases studied, it w as m uch deeper ju s t b e n e a th th e p eric h o n d riu m w h e re the cartilag e row s w e re prolonged. H e re calcification, in exceptional rases, extended for 1,000 m icrons b etw e en th e row s, b u t th e den sity of m ineral deposition w as less and th e process w a s usually n o t co n ­ tinuous from the v e rte b ra l m a rg in of Zone 4. T h e re aso n fo r this rxtended distribu tion of calcified m a trix is u n k no w n. P ossibly it adds stren g th to the rib in the region w h e re it occurs. 5. Zone of Cartilage Removal— Zone 4 w a s b o rd e re d on its v e r te b ra l m argin by a series of sac-like s tru c tu re s w hich fo rm e d th e zone of cartilage rem oval (Fig. 7). Zone 5 w as 50-75 m icron s deep. T h e saclike s tru c tu re s w e re variable in size and ex tende d in th e d irectio n of the long axis of the bone. T h e y w e re form ed by th e union of tw o or more cartilag e cell lacunae follow ing th e d e stru c tio n of th e cell ca p ­ sules by the em bryonic m arro w . T h e m a rr o w elem en ts w e re slo w er in rlestroying the calcified substance, so t h a t m a r r o w could be seen ad ­ vancing from the diaphysis to w a rd the v e n tra l end of th e rib in th e form of n a r ro w to n g u es betw e en th e walls of calcified in te rs titia l su b ­ stance which acted as guides in the process. T h e sac-like s tru c tu re s ivere bordered, th erefore, on e ith er side by calcified m atrix , while th e ir vertebral ends w e re exposed to em bryonic m a rro w . T h e y m a y be partially filled by d e g e n e ra tin g cartilage cells and by m a r r o w elem en ts (Figs. 7 and 8). 6. Zone of Ossification—Exclusive of Zone 1, which varied in d ep th depending upon the m an n er of dissecting th e rib from th e stern u m , the ventral-epiphyseal end of th e rib av e rag e d a b o u t 1,350 m icro ns (w ith variations from 1.150 to 1,450 m icrons) in depth in th e axial r e ­ gion of the bone and ab ou t 2,100 m icrons (w ith varia tio n s from 1,150 to 1,350 m icrons) in th e peripheral region. As previously m entioned, vas:ular bundles extended from Zone 1 th ro u g h th e re m a in d e r of th e epi­ physis and appeared to anasto m o se w ith th e vascu lar system in the liaphysis. Zone 6 co n stituted the b eg in ning of the diaphysis. T h e zone usually dad a convex outline on its v en tra l m a rg in (Fig. 11). E m b ry o n ic m a r ­ row w as observed advan cin g from th e diaphysis t o w a r d th e v e n tra l end of th e bone. In advance of this, ery th ro c y te s , fibrin and so m etim e s serous m aterial, and occasional endothelial cells w e re fre q u e n tly ob­ served, closely followed by larg e n u m b e rs of em bryon ic connective tissue cells (Fig. 7). D e g e n e ra tin g ca rtila g e cells freq u e n tly appe are d to be revived at this point, because of a new source of n u tritiv e m a t e ­ rial from the diaphysis. Calcified tissues resisted rem oval by th e m a rro w . A b o u t 50 m icron s vertebral to Zone 5, cells w e re occasionally seen sim ilar in size to th e primitive w a n d e rin g cells of th e em bryonic connective tissue lining th e PATHOLOGY OF PICKETS IN DAIRY CALVES persistin g bars of calcified m atrix (Figs. 7 and 13). These were osteo­ blasts, the cells associated w ith the production of acidophilic layers of osteoid tissue occasionally observed on these trabeculae. Osteoblasts averag ed about 16 microns in diam eter and were mononuclear. The nucleus m easured about seven microns in diam eter and contained one or m ore large nucleoli eccentrically placed near a pale attraction sphere. I h e cytoplasm was reticular, intensely basic, and contained small vac­ uoles. T h e calcified bars of m atrix appeared to serve at least three purposes. T hey stren g th en e d the area in which they occurred, served as guides for the advancing m arro w in cartilage removal, and formed bases for the deposition of osteoid tissue. M ost of these bars p e r­ sisted into the diaphysis to about 700 microns vertebral from Zone 5 and then m any of them w ere gradually removed by osteoclasts. O steo­ clasts appeared to function specifically in the removal of calcified cartilage and osseous tissue, and in this w ay aided in the advancement of the em bryonic m arro w and at the same time assisted in the internal reconstruction of bone. Osteoclasts w ere m ost noticeable on the v erte­ bral ends of persisting bars of calcified m atrix w here they caused t e r ­ minal erosion of the bars (Figs. 5, 15, and 16). These cells, however, were also observed about as far ventrally in the rib as w ere osteoblasts. Osteoclasts w ere variable in size and contained from two to 25 or more nuclei. Each nucleus was about five microns in diam eter and contained several coarse granules. The cytoplasm was vacuolated, took a less basic stain th an th a t of the osteoblast, and frequently showed pseudopodal projections. The means by which osteoclasts erode calcified m atrix and bone have not been included in this study. W h e th e r the process of erosion is one of osteolysis or of phagocytosis is a debated question. P ers is tin g trabeculae of calcified tissue eventually lost their cal­ cified cartilage cores by chondrolysis, and the rem aining trabeculae of bone became thicker fu rth e r in the diaphysis by peripheral additions of osteoid tissue. The presence of n arrow margins of osteoid tissue at any one time indicate early ossification in the normal rib. The line of dem arcatio n betw een osseous and osteoid tissues was a gradual one m arked by an increase in the num ber of basophilic granules so th at the stippled effect gradually became more dense in passing from osteoid to osseous tissue. These granules sometimes appeared to be deposited in striations (Fig. 5). Osteoid tissue was always accompanied by osteo­ blasts but the converse was not always true. Frequently m any osteo­ blasts, instead of a single layer, w ere seen bordering trabeculae, and whole clumps of these cells occurred in crotch-like areas where two trabeculae had united (Fig. 14). Individual osteoblasts were commonly noticed in the process of .being surrounded by osteoid tissue (Fig. 13). A fte r incorporation in the bone these osteoblasts gradually shrank and assum ed the flattened character of osteocytes (bone cells). In stained preparatio ns the osseous m aterial in the trabeculae exhibited various shades of basophilia. This fact has suggested the possibility t h a t different degrees of calcification w ere represented in the bone. D ecrease in the num ber of persisting trabeculae was accompanied by larg e r m arro w spaces betw een the trabeculae. A t about 15 mm. v erteb ral from Zone 5 the embryonic m arrow had disappeared and hemopoietic foci, along with vacuoles (probably of fatty origin) a p ­ LO M ICH IGA N T E C H N IC A L B U L L E T IN NO. 150 peared and ev entually com pletely m a sk e d th e loose connective tissue f u rth e r in th e diaphysis. H em o c y to b la sts, ery th ro b la s ts, a n d n e u t r o ­ phil m y elo cytes co n s titu te d th e m a jo rity of th e cells. M a n y sm all foci of lym phoid cells w e re s c a tte re d in th e red m a rro w . H e r e o s teo b lasts and osteoid tissue, as well as osteoclasts, w e re lim ited la rg e ly to t h e p erip heral re gio ns of the diaphysis on tra b e c u la e closely b e n e a th th e periosteum . 7. Zone of Compact Substance—T w o questions a re considered u n d e r this zone which has been called, for w a n t of a b e t t e r nam e, th e zone of com pact substance. F irs t, w h a t accounts for th e n a r r o w i n g of th e rib bone in p assing fro m th e ventral-ep ip h y sea l end to w a r d th e v e r te b ra l end? A nd second, w h a t so rt of co v ering does th e rib p ossess to sep ­ a r a t e it from su rro u n d in g tissu es? T h e cartilageno us end of th e rib w as covered by a m o d e ra te ly dense connective tissue (the p erich ond rium ) w hich v arie d fro m 150 to 500 m icrons in thickness. A t th e ju nctio n of Zones 1 and 2 th e p e ric h o n ­ drium usually exten ded as a less dense connective tissu e in w a rd to a distance som etim es as g r e a t as 800 m icrons. W h e n view ed in one plane, as in a section, it g ave th e im pression of a cone w ith th e ap e x direc ted to w a rd th e c e n te r of th e bone (Fig. 11). Blood vessels fre q u e n tly covered the crest as well as th e lateral b o rd e rs of this area. T h e p e r i ­ chondrium extended over th e zones of cell p ro life ra tio n and cell g r o w t h as a denser connective tissue of 200-300 m icron s in th ickness. B e ­ ginning w ith Zone 5 it continued as th e perio steu m . R a re ly , a n a r r o w strip w ithin the perich on driu m over Zone 4 ap p e a re d to be calcified. A t 1.5 mm. v erte b ral from Zone 5 th e p e rio s te u m th ick en e d by e x te n d ­ ing as a looser connective tissu e in w ard b e tw e e n th e p e r s is tin g t r a b e ­ culae to a distance of 1.5-2.0 mm. and th e n g ra d u a lly d ecrea sed to 150 m icrons o r less a t 25 mm. v e rte b ra l fro m Zone 5 w h e re it only ex ten d e d in b etw e en a few of th e p erip heral trabeculae. T h is d ec rease in th ic k ­ ness of th e p erio steu m o ccu rred a t a p o in t of in cre a s in g size of p e r i­ ph eral trabeculae, which la te r g re w th ic k e r and assum ed th e c h a ra c te r of a m o re com pact m a teria l th a n the tra b e c u la e in th e m a r r o w cavity. Gross m ea su re m e n ts of m id -fro n ta l sections indicate t h a t th e rib was w idest s o m ew h e re n e a r th e jun ction of Zones 5 and 6 w h e re it varied from 20 to 30 mm. in w id th in n o rm a l calves of th e ag es u n d er consideration. T h ro u g h a process of n a rro w in g , so m etim e s re f e r r e d to as tubulation, the rib g ra d u ally decreased to a m in im u m w id th of 4-8 mm. a t a b o u t 50 mm. v e rte b ra l from Zone 5. T his tu b u la tio n o c­ cu rred by term in a l erosion of th e p e rs is tin g tra b e c u la e of b o n e w hich occur in the e x tre m e p eriph eral areas and w hich ex ten d parallel to th e long axis of th e bone and th e re fo re join th e p e rio s te u m a t an oblique angle. Evidence of this erosion was t h a t one .or m ore o s te o cla sts w e re observed adjac en t to th e v e rte b ra l ends of th e se tra b e c u la e (F ig. 6). This erosion, w hich in all probability w e a k e n e d the bone, w a s c o m p e n ­ sated by th e extension of th e p e rio steu m into th e in te rio r of th e bone in the m a n n e r described above. The Costochondral Junction in Rickets This description is based on m ate ria l fro m 11 calves w h ich w e re suffering from vitam in D deficiency and w hich w ere, except fo r one anim al which died from com plications of ric k e ts a t 520 days, b e tw e e n PATHOLOGY OF RICKETS IN DAIRY CALVES 11 151 and 330 days of age at the time of slaughter. The duration of the ricketic condition varied from 38 to 239 days. The cases are about equally divided betw een (a) low blood plasma calcium rickets and (b) low blood plasm a calcium-low inorganic phosphorus rickets. No cases of low plasm a inorganic phosphorus rickets, uncomplicated by low calcium, are included in this study. The alterations which w ere observed in the ricketic bones will be considered according to the zones in the norm al rib. The histological sections will be a rran g e d in sequence of events as they occurred du r­ ing the developm ent of the ricketic rib from the normal rib. 1. Zone of Resting Cartilage—This zone usually term inated in a m od erately concave m ann er on its vertebral side similar to th a t in the n orm al rib. In prolonged rickets it varied to the ex ten t of m eeting Zone 2 in a convex m anner. In the cases of prolonged rickets there was an increase in the num ber of oval-shaped cavities apparently formed by the union of two lacunae, irregularly arranged adjacent to Zone 2 and similar to those noted in the normal bone (Figs. 17 and 18). This alteratio n was m ost pronounced in severe rickets when it involved as m uch as th e last tw o mm. adjacent to Zone 2. In rickets of shorter duration, these enlarged cavities appeared only in isolated patches adjacent to Zone 2. In such areas the m atrix betw een isogenous pairs of cells was reduced to a thin network. The m ature cartilage cells were few er in num ber and the impression was obtained th at the decrease in the ra te of g ro w th had resulted in the accumulation of im m ature cartilage cells. 2. Zone of Cell Proliferation—At the junction w ith Zone 1, the o u t­ line of this area a t first became m oderately undulated. As the severity of rickets increased, this general outline became more convex on its ven tra l m argin in th e axial region of the bone, w hereas in prolonged disease it even extended into the resting cartilage in the axial p a rt of the rib in a wredge-shape manner. The apex of the wedge was som e­ times r a th e r broad, while the sides extended out from the base tow ard the peripheral p a rt of the bone w here they either term inated in the usual m an n er or curved ventrally for some distance just beneath the perichondrium . In addition to the altered outline, Zone 2 revealed an irregular decrease in depth in rickets and was as shallow as 100 microns in certain places while of norm al dimensions in others. There was some evidence t h a t the average num ber of cells per isogenous group was reduced in rickets (Fig. 17). 3. Zone of Cell Growth—This area followed the outline of the previous zone in the same bone. I t w as unaffected in depth, except in ra re instances when it was partially obliterated in places on its verte b ral side by sinusoidal cavities containing embryonic m arrow (Fig. 17). 4. Zone of Mature Cells—W h ereas the changes thus far noted in the cartilagcnous end of the rib w ere not always conspicuous, especially in mild rickets of short duration, the alterations in Zone 4 were more pronounced and w ere therefore nun e consistently obseivcd. th e s e chaimes m ay be followed with considerable satisfaction when the se­ quence of events is traced in the costochondral junctions of the 12 M ICH IGA N T E C H N IC A L B U L L E T IN NO. 150 ricketic ribs w h e n th e y are em pirically a r ra n g e d in ord e r of in c re a s ­ ing severity of rickets. R e ta rd e d provisional calcification w as th e a lte ra tio n first observed (Fig. 9). H ere, Zone 4 was of n orm al dep th b u t th e a r e a of provisional calcification extended irre g u la rly into th e m atrix , fro m th e v e r te b ra l side of this zone, and av e rag e d less th a n 50 m icrons in depth , while in places it m easu red zero w h e n it failed to ex ten d v e n tra lly bey o n d th e fifth zone. As th e sev erity of rick e ts increased, o nly occasional longitudinal walls of cartilag e m a tr ix b etw e en the ro w s of m a t u r e cells w e re calcified. In m ore advanced rick e ts calcification w a s en tire ly ab se n t in this are a and frequ en tly only involved a rela tiv e ly small am o u n t of the m a trix in Zone 5 ad jac en t to Zone 4. L ac k of calcification in Zone 4 w as soon followed by le n g th e n in g of the ro w s of m a tu re cartilage cells. A t first, c a rtila g e ton g u es, each co n­ sisting of a g ro u p of cell row s and th e ir in te rv e n in g m atrix , p ro je c te d v erte b rally for a sh o rt distance into Zone 5 and g ave an u n d u la tin g outline of the v erte b ral m a rg in of Zone 4 (Fig. 10). L e n g t h e n in g of th e row s becam e m o re generalized in m ore advanced ric k e ts and this zone becam e deeper across the entire w idth of th e bone (F ig. 19). T h e n u m b er of m a tu re cells in row s w as g r e a t e r in p rolo n g ed rick e ts w h e n this zone reached a depth as g r e a t as 800 m icrons in th e axial region of the bone and as g r e a t as six mm. in th e m o re p erip h e ral p a r t of th e bone (Fig. 20). T h e m a trix b etw e en these le n g th en e d colum ns of cells, while hom ogeneous in appearance and free of g ra n u le s of calcified su b ­ stance, tend ed to w a rd a deeper basic stain th a n t h a t in Zone 3. T h e m a tu re cartilage cells in this zone in prolo nged rick e ts av e ra g e d less t h a n 20 m icrons in diam eter. This decrease in cell size w a s prob ab ly a result of pressure, e x a g g e ra te d by d y stro phic conditions a risin g from the increase in size of th e v e g e ta tiv e c a rtila g e mass. S everal stag es of d e g e n era tio n w ere observed and cells w e re dislodged from m an y lacunae. C artilag e cell re juv en ation , how ever, w a s co m m o n ad jac en t to Zone 5 (Fig. 22). Zone 4 followed on its v en tra l side th e o utline of Zone 3. On its v erte b ral m a rg in tw o factors helped to p ro du ce an e x tre m e ly i r r e g ­ ular outline a t the jun ction of Zones 4 and 5. One of th ese w a s th e cro w ding of th e v e rte b ra l ends of the lo n gitu din al row s of cells which caused w hole to n g u e s of cartilag e to bend so t h a t the ro w s of cells in these to ng ues no lon ger extended parallel to th e lon g axis o f th e bone. This bu ckling of cell row s w as favored by th e lack of provisio nal cal­ cification (Figs. 21 and 22). T he second factor, w hich o p e ra te d to p r o ­ duce an irre g u la r outline at the junction of Zones 4 and 5, w as the uneven ad vancem en t of the em bryonic m a r r o w from th e diaphysis (Fig. 12). Zones 2, 3, and 4 in the ricketic bone m ay c o n trib u te as m uch as tw o mm. in the axial area, and as m uch as seven mm. in th e p erip heral region, to the leng th of the rib. This thick ene d c a rtila g e a t the v en tra l end of the rib was trav e rsed , as in th e no rm al bone, by v asc u la r bundles w hich appeared to a n a sto m o se w ith vessels in th e diaphysis. 5. Zone of Cartilage Removal—T his zone pro b a b ly sh ow ed m o re pronounced alteratio n s th a n any o th e r p a r t of th e bone in rick e ts (Figs. —1 and 23). In eai 1) lickcts, cuililage icm o v al con tin u ed at a relativ ely more rapid ra te than provisional calcification so th a t ca rtila g e removal, PATHOLOGY OF RICKETS IN DAIRY CALVES 13 while occurring in the usual manner, advanced ventrally to a position ab re a st with, but never ventral to, the line of provisional calcification. A pparently the embryonic m arrow does not normally remove cartilage w hen the m atrix betw een the cells is uncalcified, which accounted for the accum ulation of m ature cartilage cells in Zone 4. In m ore severe rickets there was a failure of calcification in Zone 4 and also in m ost of the m atrix in Zone 5. The cessation of normal cartilage rem oval halted growth-involving ossification. Such cessation in grow th, however, was in some w ay compensated, as illustrated by h eig h t-a t-w ith e rs m easurem ents of severely ricketic calves which con­ tinued to gain in heig h t although at a reduced rate, even when body w eight was stationai'y or decreasing. The rib shared in this skeletal g r o w th in rickets. At least partial compensation for lack of calcifica­ tion occurred as fo llo w s : In rickets of long duration the rows of m a ­ tu re cartilage cells lengthened appreciably, which sometimes am ounted to as much as six mm., and a new m ethod of cartilage removal was apparent. The embryonic m arrow which advanced ventrally from the diaphysis was still the active agent of cartilage removal, b u t instead of p ro gressing evenly in n a rro w tongues, it was unguided by cal­ cified walls of m atrix and advanced unevenly and in a m ore or less oblique m an n er in broad tongues into Zone 4. This advancem ent de­ stroyed wide areas of cartilage, including cells and m atrix—both of which appeared to be destroyed with equal ease and rapidity (Figs. 12 and 20). E xtrem e ly large and irregularly shaped sac-like structures occurred adiacent to Zone 4. These contained in addition to the ele­ m ents seen in norm al sac-like areas, num erous erythroblasts and hemocytoblasts enmeshed in loose connective tissue (Figs. 9, 12, and 20). Osteoblasts w ere observed in advance of osteoclasts, generally, although the reverse som etimes occurred (Fig. 9). The cartilage m atrix which partially surrounded the sac-like areas assumed an acidophilic reaction. The m a rro w invariably advanced fu rth e r ventrallv, sometimes by as m uch as four mm., in the axial region of the bone (Fig. 12). The g reat unevenness of the m a rro w advancement gave the appearance of w hat has been referred to as “irregular thickening of the cartilage” and was occasionally exagg erated by actual splitting of the vertebral p art of Zone 5 (Fig. 21). Irre g u la r advancem ent of m arrow led in some places to sac-like areas of embryonic bone m arrow which appeared, in one plane as in a section, to be isolated in the zone of m ature cells (Fig. 20), and in o th er places it led to isolated masses of cartilage in the diaphysis. J u s t v erteb ral to the zone of sac-like areas, th ere occurred patches of connective tissue in which the central parts had taken a deeper acidophilic stain identical with th a t of osteoid tissue (Figs. 23 and 24). Im bedded in such tissue w ere one or several cells th a t resembled osteo­ blasts, su ggestin g th a t certain of the local hemocytoblasts had differ­ entiated into osteoblasts which had, in turn, elaborated osteoid tissue. These new patches of osteoid tissue served as bases for additional osteoblast activity. Occasionally, isolated masses of cartilage with acidophilic m a trix appeared in which the cells had not only been r e ­ juvenated but had also been transform ed to osteoblast-like cells, sug­ g e stin g th e possibility th a t cartilage can be changed en masse into osteoid tissue. Isogenous pairs of cells were observed in the larger masses of cartilage along with degenerating cartilage cells. The net 14 M ICH IGA N T E C H N IC A L B U L L E T IN NO. ISO re su lt of all of this cell activ ity w a s a zone co n sistin g larg e ly of osteoid tissue. B ecause of th e m a n n e r in w hich th e o steob lasts a p p e a re d to develop, th e ch a ra c te ris tic a r r a n g e m e n t of th e s e cells b o r d e r in g on trabeculae, as revealed in n o rm a l ribs, w a s inconspicuous in spite of the la rg e a m o u n t of osteoid tissu e t h a t w as p re s e n t (F ig. 23). T h e osteoblasts in this re g ion of th e bone have been called “O s te o id o b la s ts ” by some w o rk e rs on th e basis t h a t th ese cells prod uce tissu e w hich does n o t ossify and th ere fo re , th e cells were* r e g a rd e d as d e g e n e ra te form s of osteoblasts. Such a distinction is unjustified in th is s tu d y because th ese cells ap peared to be identical in th e i r a d u lt fo rm w ith the osteoblasts in n orm al ribs. I t does n o t seem illogical, th e re fo re , to ascribe th e failure of ossification in ric k e ts to an y one of seve ral causes — such as a deficiency in v itam in D — r a th e r t h a n to an in h e re n t d efect in th e osteoblasts. O steoclasts w e re few in n u m b e r in this region. T h e y seem ed to have no specific function in this a r e a b u t ap p e are d because of th e ir p ro x im ity to th e diaphysis. T h e osteoid tissue varied considerably in d ep th in any one bone b e ­ cause of irre g u la r m a rr o w adv an cem ent, b u t it m e a s u re d as m uch as six mm. in dep th in m ore p rolo ng ed rickets. In m ilder rick e ts th e n o r ­ m al and abno rm al types of cartila g e rem o v a l w e re ob served in th e sam e bone (F ig . 19). T h e v e rte b ra l outline of Zone 5 w as stro n g ly concave w h e re it m e rg e d into Zone 6. This tra n s itio n in zones w a s a g ra d u a l one in w hich b a s o ­ philic g ra n ules appeared in isolated areas and s u rro u n d e d individual cells in osteoid tissue. Such cells w e re conspicuous b ecause of th e c h a ra c te ristic halo abo u t each one (Fig. 23). T h e halo bec am e n a r r o w e r w ith increase in th e n u m b er of g ra n u le s and in creased ossification n e a r e r the diaphysis. T h e re also appeared small tra b e c u la e of bone, su rro u n d e d on all sides by b road layers of osteoid tissue. O steo b lasts soon b o rd e re d these trab e cu lae in th e usual m a n n e r as Zone 6 w as approached. 6. Zone of Ossification—R e o rg a n iz a tio n of m a te ria l b e tw e e n th e patches of osteoid tissue occurred soon a f te r th e first signs of ossifica­ tion w e re observed, and it w as h ere t h a t Zone 6 b e g a n (Fig. 23). P r o ­ ceeding vertebrally, th e spaces b etw e en tra b e c u la e enlarge, and th e em bryonic m a rr o w w as soon replaced by loose connective tissue. S ev ­ eral m illim eters v e rte b ra l to Zone 5, h em o po ietic elem ents' and vacuoles began to m ask the loose connective tissue in th e m a r r o w spaces. N e u ­ trophil m yelocytes w e re as n u m ero u s h ere as in th e n o rm a l re d 'm arrow . In th e e x tre m e v e n tra l are a of Zone 6 th e tra b e c u la e w e re s h o r t an d stubby and w e re b o rd e red by b ro a d osteoid tissu e m argins. T h e y also revealed fre q u e n t tra n s v e rs e a n a sto m o ses (Fig. 20). M o re v e rte b ra lly in th e diaphysis th ese trab e cu lae a s su m ed a m o re n o rm a l shape and distribution as the osteoid tissue m arg in s g ra d u a lly b ecam e n a r ro w e r . In the red m a rr o w region, trab e cu lae con tin ued to sh o w ab n o rm a lly broad m arg in s of osteoid tissue, w hich som etim e s ap p e a re d to be more sharply d e m arcate d from osseous tissue in rick e ts t h a n in n o rm a l bone. Chondrolysis w as an u n im p o rta n t g r o w t h f e a tu re h e re b ecause the ap pearance of calcified c a rtila g e cores w a s in v erse ly p ro p o rtio n a l to th e sev e rity of rickets. 7. Zone of Compact Substance— A t th e ju n ctio n of Zones 1 and 2 the perichondrium was thickened and som etim es ex ten d e d in w a rd as PATHOLOGY OF RICKETS IN DAIRY CALVES IS far as 2.5 mm. I t continued in a m oderately thickened form over Zones 2, 3, and 4 and in this location trabeculae of bone, m ediary betw een the ex ternal and internal surfaces, were usually seen. These trabeculae appeared to have the same origin as those in the v ertebral p a r t of Zone 5. T he reo rgan ization of the m aterial betw een trabeculae oc­ curred in a similar m an ner and resulted in the form ation of m arro w spaces which continued fu rth e r vertebrally as a p a rt of th e m arrow cavity. T he perichondrium continued as periosteum of about normal thickness. Tubulation appeared to occur in the norm al manner. The beading or enlargem ent of the ventral end of the rib, which was commonly observed in gross a t the tim e of slaughter, was an inconspicuous fea­ tu re of the bone w hen studied w ith the microscope. Gross m easu re­ m ents of m id-frontal sections showed th a t the diam eter of ricketic ribs varied from 20 to 35 mm. w hereas the range of norm al bones was from 20 to 30 mm. The average diam eter was about six mm. g re a te r in the ricketic bone. Microscopic observations showed t h a t the g r e a t­ est diam eter was th ro u g h the area of osteoid tissue in Zone 5, in the region ju st v erteb ral to th e cartilagenous end of the rib. An excess of osteoid tissue appeared to be the cause of this increase in size of the rib end. This enlarg em en t was more apparent than real, however, because the cu rvature of the medial border of the ventral end of the rib was g re a te r in rickets Figs. 25, 26, 27, and 28). The curvature of the lateral border increased in the same direction. This change in c u r­ v a tu re was g re a te s t at about 20 mm. vertebral to Zone 1 and could be the result of a w eakened rib responding to norm al physiological de­ mands such as respiration a n d /o r support of the diaphragm. This in­ w a rd curving of th e ven tral end of the rib exagg erated any enlarge­ m e n t t h a t did occur, especially when viewed from the medial border of the bone. The Costochondral Junction in Healing Rickets Calf C-224 was the only animal included in this phase of the study. This calf developed low plasma calcium rickets at 110 days of age. T he daily am oun t of whole milk fed was increased at a later date, and a t 140 days of age the plasma calcium began to increase and was nearly norm al when th e animal was killed at 163 days of age (Table 6). This case is of in terest because it appeared to resemble the epiphyseal type of healing rickets usually observed in the rat. This was best illus­ tr a te d in the ro en tg e n o g ram which showed at about six mm. vertebral to the cartilagenous end of the bone, a n arro w region extending half w ay across the bone from the medial side and which also showed a lack of ossification (Fig. 26). Histologically, a more or less normal costo­ chondral junction was observed except in the n arro w uncaldfied area in Zone 6, w here the trabeculae w ere small and scattered in an area consisting m ostly of connective tissue. Roentgenographic Appearance of Normal and Ricketic Ribs R o en tg en o g ra m s of norm al ribs showed a sharp line of demarcation a t the junction of the diaphysis w ith the cartilagenous end of the bone (Figs. 25, 26, and 28). The diaphysis usually ended in a predominately 16 MICHIGAN T E C H N IC A L B U L L E T IN NO. 150 convex m an n er w h e re it joined the cartilage. T ra b ecu lae of osseous tissue w e re conspicuous and indicated a d istrib u tio n of in o rg a n ic salts grossly identical w ith t h a t described above in th e histological s tu d y of the n orm al rib. T h e v en tral-ep ip hyseal end of th e rib curv ed in a medial direction. T h e walls of th e diaphysis becam e th ic k e r in p r o ­ ceeding v erte b rally fro m th e costochondral junction. Osteoid tissue has th e sam e hom ogen eou s ap p e ara n ce as c a rtila g e in th e ro e n tg e n o g ra m so t h a t ce rtain definite a b n o rm a lities w e re visible in rickets. T h e line of ju nction of c a rtila g e and diaphysis w a s i r r e g ­ ular and indefinite, and consisted of an area, v a r y in g in depth, in w hich incom plete calcification of tissues had occurred. T h e so-called “ cup­ ping,” or d evelopm ent of a concave outline of th e v e n tra l end of th e diaphysis, w as n o t a cha rac te ristic fe a tu re in th e rick e tic calf rib in the specimens studied. In m an y cases, th e end of th e diaphysis b ec am e m ore convex in rickets, due to the ad v a n cem en t of em bryonic m a r r o w in the axial region of th e bone. O th e r t h a n an increase in medial c u rv a tu re of the rib, th e ro e n tg e n o grap hic evidences of rick e ts in th e specimens studied w e re confined larg ely to the are a of excess osteoid tissue which s e p a ra te d th e v e n tra l end of th e diaphysis from the ca rtila gen ous end of th e bone. T h e im ­ po rtan c e of this localization of ricketic chang es is discussed below. Growth As a Modifying Factor in Rickets T h e effect of g r o w th upon rickets is illu stra te d w h e n calves C-237 and C-238 are com pared (Tables 8 and 9, Figs. 19 and 20). T h e se a n i­ mals received ab ou t th e same am o u n t of v itam in D in th e fo rm of w hole milk, and bo th developed low plasm a calcium rick ets by 90 days of age. T h ese calves w e re s la u g h tere d a t ab ou t th e sam e age. T h e r a te of g ain in body w e ig h t w as m uch slow er for C-237. H isto log ical studies show very definitely t h a t th e ricketic a lteratio n s in th e rib w e re m u ch less extensive in the slow er g ro w in g calf. A com parison of calves C-170 and C-232 m ay be used to illu s tra te the influence of ag e in rickets (Tables 5 and 7, Figs. 10 and 29). C-232 developed low plasm a calcium rickets by 100 days of age, exhibited low in organic ph osp ho rus values at 140 days, and w as s la u g h te re d at 161 days of age. C-170 developed low p lasm a calcium ric k e ts by 170 days of age, th e in org anic p hosphorus fluctuated c o n s id e ra b ly ' a t this tim e b u t w as n ot definitely s u bno rm al until a f te r 280 days of age. This anim al w as s la u g h tere d a t 330 days of age. B o th calves gain ed subnorm ally in body w eight. K eepin g in mind the ag e difference an d th e fact t h a t the older calf had rickets for 160 days, w hile th e y o u n g e r calf had ab out th e sam e severity of rick e ts for only 61 days, as ju d g ed by the blood plasm a analyses, it is significant t h a t in ro e n tg e n o graphical as well as in histological studies, th e y o u n g e r calf (C-232) m anifested th e m ore ex tensive ricketic alteratio ns, alth o u g h it s u f­ fered from the disease fo r a m uch s h o rte r time. Complications in Rickets Calf C-188 (T able 5) developed rick e ts a t 380 days of ag e and in addition to the usual sy m p tom s the anim al show ed muscle a t r o p h y and e x tre m e em aciation. C-188 died at 520 days of age, follow ing a c o m a ­ PATHOLOGY OF RICKETS IN DAIRY CALVES 17 tose condition and failure to respond to subcutaneous injections of viosterol and intravenous injections of calcium gluconate and m a g ­ nesium sulphate solutions. I t lost more than a pound of body w eight daily during the last 100 days of life. On post m o rtem examination, th e m arked pitting and erosion of the artic u la tin g surfaces w ere m ost conspicuous, particularly at the distal end of the fem ur and the proximal end of the hum erus (Fig. 33). This condition existed in th e peripheral as well as the central p a rts of the articular areas. W h e re the cartilage surface was not destroyed, it was v ery loose and easily removed by the lingers. Bone of a porous-plate appearance w as exposed. Spongy pads of protein-like material covered these areas. R o entg en o g ra m s of thin sections of bone removed from such areas showed an irregular rarefaction of the bone beneath the eroded or pitted surfaces (Fig. 32). Histological studies indicate th a t fibrous connective tissue occupied these rarefied, calcium-free areas. In the ven tral end of the rib a few faint indications of arrestedg r o w th lines, m ark in g areas of increased num bers of transverse trab e c­ ulae, w ere observed in the ro e n tgen ogram vertebral to the cartilagen­ ous end of the rib (Fig. 25). Histologically, provisional calcification w as som ew hat re ta rd ed and incomplete. A shallow area of calciumfree osteoid tissue was vertebral to Zone 4. Following this area were sh o rt trabeculae of bone with broad margins of osteoid tissue and more th a n th e norm al num ber of transverse anastomoses. T h e lack of ex­ tensive changes in this ricketic rib was attribu ted partly to the age of th e animal, and p artly to the severity of the disease and its halting effect upon grow th. , Type of Rickets and Histological Alterations W h en observed in prepared sections, the histological alterations in th e rib appear to be identical in low plasma calcium rickets and in rickets associated with low calcium and low inorganic phosphorus. DISCUSSION R ickets in dairy calves suffering from a deficiency of vitam in D was ch aracterized by changes in the bones (Figs. 1-34), which were ap ­ p aren tly accompanied by decreased concentrations of the plasma cal­ cium a n d / o r inorganic phosphorus (Tables 2-9). Other changes, more inconsistently found, w ere the accumulation of bile in the gall bladders and the presence of bile stained ingesta in the upper region of the small intestine. The costochondral junction at the ventral end of the rib w as selected as the best and most convenient index to the degree of rickets in calves (Figs. 1 and 12). W h e n com pared w ith the norm al cases, the ricketic costochondral junctions w ere characterized fundamentally by retarded and incom­ plete provisional calcification of the cartilage matrix, which in turn caused te m p o ra ry failure of cartilage removal by the embryonic m a r­ ro w (Figs. 9 and 10). M ature cartilage cells then accumulated up to a point w here a ricketic type of cartilage removal was initiated by the em bryonic m arrow . It was this la tte r process which caused the most conspicuous alteratio ns in the rib (Figs. 12, 19, 20, 21, and 29). 8 M ICH IG A N T E C H N IC A L B U L L E T IN NO. 150 I f th e th e o ry is accepted t h a t r e ta rd e d provisional calcification is the fu n d a m e n tal ch a n g e in th e bone in ric k e ts — and t h e r e is e v e iy reason to believe t h a t such is th e case in ric k e ts caused b y a v ita m in D deficiency— th e n th e histological alte ra tio n s in th e rick e tic b o nes m ay be explained in w h a t app e ars to be a v e ry s a tis fa c to ry m a n n e r. Thus, in th e absence of th e lo ngitudinal trab e cu lae of calcified c a rtila g e m a ­ trix to act as guides, the em bryonic m a rr o w in th e rick e tic ty p e of c a r­ tilage rem oval advanced irre g u la rly and obliquely in b ro a d to n g u e s which caused an uneven decrease in d ep th of th e zone of m a t u r e cells (Fig. 12). T h e irre g u la r m a n n e r of m a rr o w ad v a n c e m e n t led to sac-like areas which appeared, in one plane as in a section, to be iso lated in th e zone of m a tu re cells. In o th e r instances it re s u lted in w h a t ap p e are d to be isolated m asses of cartilage v e r te b ra l to th e zone of m a t u r e cells. V ertebrally, in th e region adjacent to th e la rg e sac-like areas, w e re seen patches of connective tissue which occupied m o s t of t h a t p a r t of th e rib (Fig. 23), In th ese p atches h em ocy to b lasts ap pe are d to ch a n g e to o s t e o ­ blasts which produced areas of osteoid tissue in th e p atch e s of fibrous connective tissue (Fig. 24). E v e n tu a lly m o s t of this zone w a s occupied by osteoid tissue, th e accum ulation of w hich w as pro b a b ly of a co m ­ p e n s a to ry n a tu re in th e absence of bone fo rm a tio n . T h us, in re a lity ca rtila g e rem oval in rick e ts w as fu n d a m e n tally th e s am e as t h a t in th e no rm al bone, b u t a different ap pearan ce w as prod uced b ecause of th e lack of provisional calcification. C hanges w e re less exten siv e f u r th e r v erte b rally in the diaphysis as th e bone ap p ro ach ed a m o re n o rm a l appearance. T h e beading or swelling com m only o bserved in th e v e n tra l ends of ribs from rick etic calves was n o t a conspicuous chang e w h e n view ed in th e microscope. T h e re was, h ow ever, a m o d e ra te sw ellin g of th e v e n tra l end of th e bone, th e origin of which ap p e are d to be due to th e accum ulation of osteoid tissue. This e n la rg e m e n t w as e x a g g e r a te d by the increased cu rv a tu re of the medial bo rd e r of th e rib (F igs. 25, 26, and 28). R ickets affected the g ro w in g ends of bones (F igs. 25, 26, and 28). The alte ra tio n s w e re less severe in th e slow er g r o w i n g calves (Figs. 19 and 20) and also in th e older anim als w h e n th e r a t e of g r o w t h had decreased (Figs. 10 and 29). U n d e r the conditions of this stu d y g r o w th occurred a t a m o re rapid r a te in the v e n tra l end of. th e rib t h a n in th e limb bones. This is also indicated in th e n e w b o rn calf w h ich is up on its feet w ithin a few ho u rs a f te r birth, w hich indicates t h a t a t b irth th e leg bones are relatively m o re m a tu r e t h a n t h e ribs. T h e localiza­ tion of the ricketic changes a t th e end of th e rib is a f u n d a m e n ta l p oin t of som e im portance. In the first place, it is add itio nal evidence t h a t rickets occurred only w h e re th e re w as g ro w th , and secondly, it has a practical application in m a k in g ash d e te rm in a tio n s on ric k e tic bones. Obviously, even th o u g h th e re m ay be a la rg e excess of o steo id tissue at th e costo cho nd ral jun ctio n it will n o t be tru ly reflected in th e ash analysis of the. entire bone since the ricketic cha n g es a re confined to a v ery small p a r t of an o th erw ise well ossified rib. F r a g m e n t a r y evidence indicates t h a t th e epiphyseal type o£ h ealin g rickets occurred in one calf in this stu dy (Fig. 26). W ith th e exception of th e blood p lasm a and possibly th e lym ph, th e changes which occurred in th e so ft tissues of th e bo d y w e r e n o n ­ PATHOLOGY OF RICKETS IN DAIRY CALVES 19 specific and w ere probably the result of general im pairm ent of bc>dy health. In one case of advanced rickets, however, muscle atroph y and erosion of the artic u la r surfaces w ere observed in addition to the usual sym ptom s of rickets in calves. P ittin g and erosion of the joints was seen in several of the ricketic calves in the present investigation but never to such a m ark e d degree as in the one calf described above (Figs. 33 and 34). H u ty ra and M arek (9) included ulcerous d estru c­ tion of the joint cartilages along w ith inflammation of the joints in their list o f general anatomical changes of rickets in cattle. They also noted t h a t severe rickets was accompanied by increasing emaciation and t h a t prognosis depended upon nervous disturbances and complica­ tions. Gullickson and co-w orkers (18)^ did not m ake histological studies of the rickets-like disease which they encountered in calves. Their g en­ eral observations, however, re g ard ing gain in body weight, composition of the blood plasm a and skeletal alterations, including erosion of the a rticu latin g surfaces, are fundam entally in agreem ent w ith the find­ ings of our w o rk on rickets in dairy calves. T he tw o types of low vitam in D rickets observed in this study ap­ pear to be histologically identical. P erhaps one should n o t expect to find histological differences in bones from cases of low blood calcium rickets and cases in which the concentrations of both calcium and inorganic phosphorus are low. A com parison of the results which were obtained in this investiga­ tion w ith the reports in the literatu re indicates th at the alterations in ricketic bone are fundam entally the same in the calf as those described for the y o u ng r a t and for the infant. This similarity in response to a deficiency of the antiricketic factor dem onstrates the suitability of the dairy calf for problems of fundamental research in the n utrition of vitam in D. SUMMARY This investigation was designed to determ ine the histological a ltera­ tions th a t occur in the tissues of dairy calves suffering from a deficiency of vitam in D. The study was based on 16 grade Holstein calves which w e re selected from a group of more than 100 animals available for this work. T he ages of the calves varied from 151 to 520 clays at the time of death. Five of these calves w ere normal, while the rem aining 11 calves represented several stages in the severity of rickets. The d ura­ tion of th e disease varied from 38 to 212 days. One case of healing rickets was included in the study and also one case of rickets in which muscle atro p h y and erosion of th e joint cartilage had occurred. L o w vitam in D rickets in dairy calves was characterized by changes in the bones which w ere apparently accompanied by decreased con­ ce n tration s of calcium a n d /o r inorganic phosphorus in the blood plasma. The costochondral junction at th e ventral end of the rib was found to be th e best index to ricketic changes in the skeleton. M id-frontal sections of about four mm. in thickness were tak en from the last three inches of the v en tral end of each rib and studied by the comparison of ro e n tg e n o g ram s, photog rap hs of specimens stained in silver n itrate solution, and histological sections. 20 M ICH IGA N T E C H N IC A L B U L L E T IN NO. 150 R o e n tg e n o g ra m s w e re su perior to th e specim ens stained in silver n i t r a t e fo r this study. Sections w e re p re p a re d fo r h istological s tu d y by decalcification in 5 p er c e n t aqueous solution of p o ta ss iu m dichrom a te followed by th e paraffin im bedding tech n iq u e an d w e r e re g r e s sively stained in hem ato x y lin and eosin solutions. D etailed descriptions w e re m ade of th e co sto cho ndral ju n c tio n s of b o th n o rm al and rick etic ribs. In the specim ens studied, th e histolo gical changes w e re largely confined to a re latively sm all p o rtio n of th e bone a t th e co sto cho ndral junction. R e ta rd e d provisional calcification of th e ca rtila g e m a t r i x ap p e a re d to be th e fu n d a m e n tal ch ang e in rickets. T h e m o st conspicuous c h a n g es in th e m icroscopical study, h ow ever, w e re th e irr e g u la r re m o v a l of ca rtila g e by the em bryonic m a rr o w an d th e a c cu m u latio n of excess osteoid tissue. T h e b ead ing of rick etic ribs ap p e are d to be cau sed b y th e ac cum ulation of osteoid tissu e an d w a s e x a g g e ra te d in a p p e a ra n c e on the m edial b o rd e r by increased c u r v a tu re of t h e rib in a m edial direction. “ C upping” of th e v entral-ep ihy seal end of th e diaphysis w as n o t a p ro m in e n t fe a tu re in ro e n tg e n o g ra m s of th e c o s to ch o n d ral ju nctions of rick etic calves used in this study. G ro w th w as an im p o rta n t m odify in g fa c to r in rick ets. M o re severe rick e ts w as associated w ith m o re rapid g ro w th . W ith in a g iv en period of time, y o u n g e r calves developed m o re florid ric k e ts t h a n did older calves. L o w plasm a calcium rick e ts appe are d to be histologically identical to low calcium-low inorganic pho sp horu s rickets. F r a g m e n t a r y evidence s u g g e s te d t h a t th e epiphyseal ty p e of h e a l­ in g rickets occurs in dairy calves. E m a c ia tio n of th e body an d erosion of the artic u la r ca rtilages occurred as com plications in one case of rickets. j H istological alte ra tio n s of rick e ts in calves, as o bserv ed in th is in ­ vestigation, ap pear to be fun d a m e n tally th e sam e as th o se re co rd e d in th e lite ra tu re for the in fan t and for t h e y o u n g rat. A b n o rm a l accum ulations of bile of an o ra n g e to y ellow color and of viscous ch a ra c te r w e re o bserved in several cases of ric k e ts a t p o s t m o rtem . Bile stained in g e s ta occasionally occ u rre d in th e u p p e r p a r t of th e small intestine. E n te ritis w as n o ted in a few cases. PATHOLOGY OF RICKETS IN DAIRY CALVES CONCLUSIONS In the specimens studied, the following conclusions seem to be ju s ­ tified : 1. A t autopsy, low vitam in D rickets in dairy calves is characterized principally by changes in the bones. These changes may be con­ veniently studied in the ventral end of the eighth rib. 2 . R etard e d provisional calcification of cartilage m atrix appears to be the fundam ental change in rickets. The most conspicuous his­ tological changes in the disease, however, are irregular removal of cartilage and accumulation of excess osteoid tissue. 3. Accum ulations of osteoid tissue are responsible for the beading of ricketic ribs. This beading is typically exaggerated in gross appearance on the medial border of the rib by increased cu rv a­ tu re of the rib in a medial direction. 4. G row th is an im p ortant modifying factor in rickets. The ricketic condition is confined laYgely to the grow ing area at the end of the bone. M ore severe rickets occurs in more rapidly growing dairy calves. Age, because of its relation to grow th, is also a m odifying factor in th a t younger calves develop more florid rickets than older calves within a given period of time when maintained under similar conditions. 5. Histological alterations of rickets in calves are fundamentally the same as those recorded in the literature for the infant and for the young rat. M ICH IG A N T E C H N IC A L B U L L E T IN NO. 150 LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Reed, O. E., and H uffm an , C. F.— F e e d in g of c o n c e n tra te s to dairy cattle. H e av y feeding of c o n c en trate s w ith o u t th e p ro p e r quality of ro u g h a g e is d etrim e n ta l to th e animal. Mich. A g r. E xp. Sta. Q u a rt. Bui., 8 : 118 (1926). H uffm an, C. F.— M ak e stu d y of rick ets in calves. S u nshin e c o u n t ­ eracts deficiencies in ration s w hich w ould cause disease. Mich. A gr. Exp. Sta. Q u a rt. Bui., 14: 42 (1931). H uffm an, C. F., and Duncan, C. W .—-Vitamin D studies in cattle. I. T h e a n tira ch itic value of h ay in th e ra tio n of d airy cattle. J. D a iry Sci., 18: 511 (1935). H uffm an, C. F., and Duncan, C. W.— V itam in D studies in cattle. II. T h e v itam in D sparing* action of m a g n e s iu m in th e ra tio n of d airy cattle. J. D a iry Sci., 18: 605 (1935). D uncan, C. W., and H uffm an, C. F.—V itam in D studies in cattle. III. Influence of solar u ltraviolet ra d ia tio n upon th e blood c h e m ­ istry and m ineral m etabolism of d airy calves. J. D a ir y Sci. (in pre ss) (1936). Bechtel, H. E., H uffm an, C. F., Duncan, C. W., and H o p p e rt, C. A.— V itam in D studies in cattle. IV. Coi*n silage as a source of vitam in D for d airy cattle. J. D a iry Sci. (in p re ss) (1936). Bechdel, S. I., L a n d sb u rg , K, G., and Hill, O. J.— R ic k e ts in calves. Pa. A gr. Exp. Sta. Bub, 291 (1933). Rupel, I. W., B o hstedt, G., and H a r t, E. B.—V ita m in D in the n u tritio n of th e d airy calf. Wis. A gr. Exp. Sta. Res. Bui. 115 (1933). H u ty ra , F., and M arek , J.— Special p ath o lo g y and th e ra p e u tic s of th e diseases of dom estic animals. 3:213-269. 3rd ed. A le x a n d e r E g e r, Chicago. (1926). M arek , J., and W ellm an, O.— Die rachitis. G u s ta v F isch er, J en a. (1931). T heiler, A.—T h e o steod ystrop hic diseases of d o m esticated anim als. Vet. J , 90: 143, 183 (1934). H a rris , H. A.— Bone g ro w th in h e a lth and disease. O x fo rd Univ. P ress, L o n don (1933). M axwell, J. P., H u, C. H., and T urnbull, H. M.—-Foetal rickets. J. P a th . Bact., 35: 419 (1932). P appenheim er, A. M.— T he anatom ical cha n g es w hich acco m p any th e h ealing of experim en tal r a t rick e ts u n d e r th e influence of cod liver oil or its derivatives. J. Exp. Med., 36: 335 (1922). Dodds, G. S.,. and Cam eron, H. C.— S tudies on e x p e rim e n ta l rick e ts in rats. Am. J. Anat., 55: 135 (1934). M axim ow , A. A., and Bloom, W .—A t e x t-b o o k of h istolo gy. W. B. S au nders Co., P hiladelphia (1930). Leriche, R., and Policard, A.— T h e n o rm a l and p a th o lo g ical ph y sio l­ ogy of bone. C. V. M osby Co., St, Louis (1928). Gullickson, T. W ., P alm er, L. S.-, an d Boyd, W . L .— A rick e ts-lik e disease in y o u n g cattle. Minn. A g r. Exp. Sta. Tech. Bui., 105 (1935). PATHOLOGY OF R ICK ETS IN DAIRY CALVES Table 1. C alf N o. A ge at death (days) 23 Calves selected for studies on bone histology. D uration of rickets (days) C -1 5 1 ............... 194 N orm al C -1 7 6 .............. 317 “ C -2 2 9 ............... 163 C -2 3 1 ............... 161 C -2 3 3 ............... 161 C -1 4 8 .............. Rem arks E xposed to sum m er sun. R eceived 5 cc. cod liver oil and 15 gm. of M gO daily. R eceived 4 lb. of irradiated m ilk daily. “ R eceived 4 lb. of irradiated m ilk daily. 232 212 A te 1 lb . sun-cured h a y daily. C -1 5 5 .............. 195 145 A te 1.5 lb. sun-cured h a y daily. C -1 7 0 .............. 326 160 R eceived 5 cc. cod liver oil daily. C -1 7 1 .............. 327 185 R ubbed 2 cc. 250D viosterol daily upon the skin. Low Ca—• low P rickets. U nusually poor gains in body w eight. R eceived 5 cc. cod liver oil daily. Received 4 lb. of irradiated m ilk daily. Low P — low Ca rickets. C -1 7 3 .............. 319 239 C -1 7 5 .............. 318 38 C -1 8 8 .............. 520 140 C -2 2 4 .............. 163 ' C -2 3 2 .............. 161 61 R eceived 20 lb. skim m ilk daily. C -2 3 7 .............. 151 61 R eceived 24 lb. w hole m ilk daily. in body w eight. C -2 3 8 .............. 161 71 R eceived 20 lb. w hole m ilk daily. 53 Low Ca rickets. Low Ca— low P rickets. Low P — low Ca rickets. R eceived 5 cc. cod liver oil and 32 gm. MgCC>3 daily. rickets. R eceived 15 lb. corn silage daily. R eceived 20 lb. w hole m ilk daily. low Ca rickets. Low Ca Low Ca— low P rickets. H ealing last 23 days from Low Ca— low P rickets. Low Ca rickets. Low Ca rickets. Poor gains M IC H IG A N TE C H N IC A L B U L L E T IN NO. 150 24 Table 2. Growth data and blood plasma analyses. B lo o d p lasm a A ge C alf N o . (days) C M 510) 3 -1 7 6 (3) ................................ Inorg. P Ca (lb.) (per cent norm al) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 19 4 (2) 85 95 105 114 122 122 126 143 163 177 193 214 231 246 253 255 253 257 276 288 85 86 87 86 84 78 74 77 82 82 83 86 87 87 84 80 76 74 76 78 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 317(2) 98 108 110 121 131 142 158 176 177 202 215 233 237 230 228 250 262 277 296 311 328 345 367 373 399 416 433 440 434 453 459 470 98 98 91 91 90 90 92 95 89 93 92 94 89 81 75 79 79 79 82 83 84 86 89 88 91 92 93 92 89 90 90 91 (0 B orn M arch 4, 1932— m ale. (2) Slaughtered. (3) B orn M a y 29, 1932— m ale. Mg (cm .) (per cen t norm al) 7 6 .8 101 1 2 .4 1 2 .3 6 .6 9 6 .1 0 2 .3 7 2 .3 0 8 1 .2 100 1 3 .5 1 3 .6 6 .9 1 7 .5 3 2 .0 9 2 .1 1 8 5 .2 99 i3 .6 1 4 .1 1 5 .4 8 .0 6 7 .4 0 8 .2 3 2 .3 4 2 .7 8 2 .7 5 9 2 .5 102 9 7 .8 102 1 4 .1 1 2 .8 7 .5 3 8 .3 9 3 .4 7 2 .3 3 9 9 .0 99 1 3 .0 1 2 .6 1 2 .8 1 3 .8 6 .0 1 8 .1 2 7 .9 6 7 .4 4 2 .4 5 2 .4 0 2 .5 7 2 .5 8 7 7 .7 101 1 3 .8 1 5 .0 8 .8 7 8 .8 3 2 .4 0 2 .4 7 8 1 .7 100 1 3 .9 1 3 .3 7 .6 2 6 .7 9 2 .3 6 2 .0 7 8 7 .2 101 (m g. per 100 cc.) 1 3 .9 7 .1 4 1 .9 8 •1 2 .9 1 3 .8 6 .7 2 8 .6 2 2 .3 7 1 .6 3 9 1 .3 99 96 9 6 1 0 .3 8 .3 3 5 .1 7 5 .4 8 2 .9 1 9 4 .3 9 5 .5 94 1 1 .4 1 0 .6 6 .6 9 7 .5 8 2 .7 4 2 .8 0 9 9 .8 95 ii.2 1 1 .8 1 1 .5 7 .7 1 6 .2 2 7 .3 5 2 .7 4 2 .6 2 3 .3 1 1 0 1 .2 94 1 0 .8 9 .9 6 .2 8 6 .3 8 2 .6 0 1 0 7 .8 98 1 0 .9 1 0 .7 1 0 .5 6 .8 7 6 .6 9 6 .2 2 2 .4 4 3 .6 8 2 .6 7 1 1 0 .2 99 1 1 .2 1 0 .8 6 .7 9 6 .4 1 3 .5 2 2 .1 7 PATHOLOGY OF RICK ETS IN DAIRY CALVES Table 3. 25 Growth data and blood plasma analyses. Blood plasm a A ge W eight H eight Calf N o, Ca (days) C -2 2 9 0 ) ............................... C-231 (-1) ............................... C -233(4) ............................... (*) (-) (■’) O) (5) B orn October Slaughtered. B orn O ctober B orn O ctober Slaughtered. (lb.) (per cent normal) 10 20 30 40 ,50 60 70 80 90 100 110 120 130 140 150 160 163(2) 80 92 100 110 129 148 165 179 197 214 233 246 256 268 284 304 312 80 83 83 83 89 94 96 96 99 99 100 99 96 94 94 96 97 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 161(») 97 106 116 126 144 163 181 192 202 210 228 239 236 237 249 264 265 97 96 96 95 99 104 106 103 101 97 98 96 89 83 82 83 83 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 161(6) 104 114 123 136 146 162 174 192 206 224 245 252 278 309 337 354 104 103 102 102 101 103 102 10.3 103 104 105 101 104 109 112 111 (per cent normal) (cm.) 7 1 .2 95 7 5 .0 96 8 1 .8 98 8 6 .7 96 8 8 .0 93 7 4 .5 ' Mg Inorg. P (mg. per 100 cc.) 1 2 .9 1 3 .8 1 3 .4 1 2 .9 1 2 .2 1 2 .3 1 2 ,0 1 2 ,3 1 3 .2 12.1 1 1 .8 12.1 8 .7 12.1 1 1 .7 1 1 .8 8 .7 4 8 .5 6 7 .9 1 8 .3 9 8 .1 2 7 .5 8 7 .0 9 6 .4 1 7 .7 7 7 .2 7 8 .0 1 9 .2 9 7 .5 8 9 .2 4 9 ,6 9 9 .1 9 2 .2 5 2 .2 1 3 .2 3 2 .5 5 2 .4 9 2 .7 2 2 .5 9 1 .9 0 2 .2 3 2 .3 2 2 .2 6 2 .4 1 2 .6 2 2 .5 5 2 .2 5 2 .9 5 101 1 2 .6 6 .1 9 2 .3 3 7 8 .2 102 8 4 .8 103 8 8 .8 100 9 4 .2 101 9 5 .6 98 1 1 .7 1 2 .6 1 2 .3 1 1 .5 1 1 .6 9 .7 12 .1 1 1 .8 1 0 .7 12 .3 1 1 .4 1 2 .7 1 1 .5 1 1 .7 10.9 6 .9 8 8 01 8 .2 2 7 .8 6 8 .2 5 8 .3 3 7 .8 6 7 .7 4 7 .0 2 8 .3 9 8 .7 4 7 .9 4 8 .6 8 9 .9 2 8 .0 6 2 .8 3 3 .1 7 2 .5 2 3 .1 7 2 .7 7 2 .4 9 2 .0 7 2 .4 5 2 .3 8 2 .9 0 2 .4 3 2 .5 8 2 .3 2 2 .9 1 3 .1 8 1 3 .4 1 1 .8 7 .5 3 8 .3 3 2 .6 9 2 .7 5 1 3 .4 12 .7 1 2 .7 1 1 .9 1 1 .7 12.1 1 2 .0 12 .3 1 2 .7 1 2 .9 1 2 .4 11.6 1 1 .8 8 .2 8 8 .5 6 7 .7 8 8.01 7 .4 0 7 .7 7 7 .2 3 7 .0 3 8 .5 0 6.31 8.01 8 .2 3 8 .5 3 2 .2 5 2 .6 1 2 .6 3 2 .6 7 2 .4 1 2 ,2 7 2 .4 5 2 .4 8 2 .9 1 2 .6 3 2 .7 5 2 .1 7 2 .3 2 7 3 .5 100 7 7 .7 101 8 1 .0 99 8 6 .2 97 9 2 .8 99 9 7 .3 99 16, 1933— m ale. 25, 1933— m ale, 28, 1933— male. P ittin g of articulating surfaces of femur and humerus. • M IC H IG A N T E C H N IC A L B U L L E T IN NO. 150 Table 4. Growth data and blood plasma analyses. B lo o d p la sm a A ge w e ig in C alf N o. (days) -1 4 8 0 ). 155 (r>) . 10 20 30 40 50 60 70 80 90 100 110 120 130 140 1.50 1 6 0 (2) 170 180 190 200 0 ) 210 220 0 ) 230 232(5) 10 20 30 40 50 60 70 80 90 100 11 0 120 1 3 0 (0 140 150 160 170 180 190 1 9 5 (8) (0 (0 C) (0 B orn M arch l , A ge 156 days. A ge 197 days. A ge 216 d ays. (lb.) (per cent norm al) 91 98 110 118 129 144 154 166 171 171 169 168 165 165 165 167 172 170 171 175 180 190 187 91 89 91 89 89 92 90 89 86 79 73 67 62 58 55 53 52 49 47 47 46 47 45 88 102 115 123 134 143 149 157 166 171 177 200 204 199 203 206 213 217 221 223 88 92 95 92 92 91 87 85 83 79 76 80 77 70 67 65 64 62 61 60 (cm .) (per cent norm al) 7 7 .5 101 8 3 .5 102 8 7 .0 Mg Inorg. P Ca (m g. per 100 cc.) 2 .4 0 2 .2 3 1 2 .5 1 1 .9 1 1 .3 1 1 .4 1 0 .1 5 .6 1 4 .4 6 5 .8 4 6 .2 2 5 .4 3 2 .6 3 2 .1 5 100 1 0 .0 1 1 .7 1 1 .8 5 .0 4 5 .3 2 4 .0 9 2 .2 8 2 .6 7 3 .3 1 8 7 .7 96 1 2 .2 1 1 .8 3 .9 5 2 .7 6 3 .0 2 2 .8 5 8 5 .8 89 3 .1 2 2 .5 8 3 .9 3 2 .6 6 1 .7 3 2 .8 0 8 4 .5 84 1 2 .0 1 0 .4 9 .7 9 .9 9 .6 2 .9 6 3 .2 3 2 .2 3 2 .1 5 1 2 .3 8 .5 7 3 5 .0 0 4 .0 5 4 .1 3 2 .1 2 1 .2 2 0 .9 9 6 .8 3 8 .2 3 7 .2 7 2 .2 9 2 .6 9 2 .8 0 D ow n 7 7 .5 103 1 3 .7 1 3 .5 1 2 .7 8 3 .7 105 1 0 .9 6 .2 8 2 .3 1 8 7 .8 103 9 .3 9 .9 9 .3 7 53 7 .6 2 8 .3 3 2 13 2 .2 4 2 .6 9 9 0 .0 100 9 .8 9 .7 7 .5 3 6 .5 4 2 .0 5 2 .4 7 9 1 .8 97 9 .3 8 7 7 .3 1 6 .3 2 2 .1 9 9 3 .7 94 8 .1 8 .4 8 .9 7 9 5 .8 1 5 .2 7 8 .5 0 7 .9 1 2 .0 7 1 .7 7 1 .6 7 1 .6 6 1932— m ale. Legs bow ed. J o in ts sw ollen . U nable to w alk to scales. U nable to sta n d lo n g enough to drink m ilk. ( 5 ) C onvulsion . Slau ghtered . («) B orn M arch 10, 1932— m ale. (») A ge 134 d ays. L igh t c o n v u lsio n . (8) Slau ghtered . PATHOLOGY OF RICKETS IN DAIRY CALVES T a b l e S. 27 G r o w t h d a t a a n d b lo o d p l a s m a a n a ly s e s . B lood plasm a A ge Calf N o. C - 1 7 0 0 ............................... C -188(6) ............................... (i) 0 0 (i) (*) (») Ca (per cent normal) (days) (lb.) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 3000 310 3200 3260 97 96 103 111 118 128 140 146 153 161 178 189 194 198 208 220 230 243 269 286 301 326 345 355 377 396 411 429 448 462 463 461 473 97 87 85 83 81 82 82 79 77 74 77 76 73 70 68 69 69 70 74 76 77 81 84 84 86 88 88 90 92 92 91 89 89 360 370(6) 380 3900 400 410 420 (s) 430 440(6) 450 460 470 480 490(16) 500 510 520(H) 500 525 524 523 549 520 538 540 532 520 529 521 509 496 466 436 406 90 93 92 91 94 88 90 90 88 85 85 82 79 76 71 66 61 Born M a y 1, 1932— m ale. G ettin g stiff in legs. B ack arched. V ery stiff in legs. Slaughtered. B o m N ovem ber 14, 1932— male. Joints enlarged. Rear legs stiff. (7) (s) (9) (1(l) (») (cm.) (per cent normal) 7 6 .2 100 Inorg. P Mg (mg. per 100 cc.) 1 1 .6 6 .6 9 • 2 .1 2 12 6 1 3 .6 5 .8 1 7 02 5 .7 4 1.99 2 .1 4 2 .6 2 13 5 14.1 7 .7 6 6 .6 5 2 74 2 88 1 3 .2 1 1 .9 6 .3 8 7 35 2 45 2 .5 1 i2 .2 1 0 .6 5 .9 5 4 90 5 .1 7 2 .6 9 1 .9 0 2 .6 3 9 6 8 .7 5 .1 0 5 .5 8 2 .0 8 2 .1 3 8 0 .2 98 8 4 .0 97 8 8 .2 96 9 0 .7 94 9 2 .7 91 9 8 .2 94 8 7 10 5 9 .5 6 .5 4 6 69 7 .5 3 2 .5 7 2 .9 3 2 .7 5 1 0 2 .7 95 8 8 9 .3 6 .1 3 5 .3 2 2 .6 0 2 .6 9 1 0 3 .7 95 8 1 7 .3 8 3 6 41 5 .7 1 5 .1 2 2 .6 2 2 .5 8 2 .3 3 1 0 7 .7 96 8 .0 9 .4 5 .3 4 4 .7 3 2 .7 8 2 .5 9 1 0 .8 9 .5 8 .3 1 0 .3 8 .6 8 .2 8 .3 7 .4 1 0.3 7 .8 8 .2 7 .9 8 .2 8 .5 8 .6 8 .0 8 .1 8 .1 2 8 .6 2 9 .1 9 5 .9 3 6 .6 2 8 .2 8 6 95 7 .2 5 5 08 4 .6 3 5 17 5 63 4 .3 7 4 19 3 29 2 96 3 .5 0 3 .1 0 3 .5 5 2 .5 6 2 .2 6 2 .90 2 .6 1 2 .5 8 2 .6 0 2 .25 2 .8 3 2 . 67 2 .45 2 .8 3 2 59 2 49 2 49 2 .3 7 1 0 9 .8 96 1 1 2 .8 98 1 1 4 .8 98 1 1 4 .2 96 1 1 3 .8 95 K nees bowed. Stands cross legged in rear. D rags rear feet. Very stiff. Lies down m ost of time. A ge 487 days. In coma, D ied. 28 M ICH IG A N T E C H N IC A L B U L L E T IN NO. 150 Table 6. Growth data and blood plasma analyses. B lo o d p la sm a A ge n e ig u i W eig h t (days) C -1 7 1 0 )1................................ (lb.) (per cent norm al) (cm .) (per cent norm al) 143 151 84 79 80 83 85 oO 83 SI 8 3 .8 97 158 161 168 73 69 67 8 5 .2 95 140 181 179 64 59 8 7 .8 92 170 180 190 280 290 300 310 (3) 320 327 (4) 181 179 186 198 210 215 219 225 230 241 257 264 269 280 284 283 283 54 51 51 53 54 54 53 53 52 53 55 55 55 56 56 54 54 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 163 (0) 93 106 117 127 143 162 177 192 213 229 244 264 283 298 312 329 342 93 96 97 95 99 103 103 103 107 106 105 106 106 105 103 104 107 10 20 30 40 50 60 70 80 90 100 110 120 210 220 240 250 260 84 87 97 110 123 7 8 .0 103 99 Mg Inorg. P Ca C alf N o. (m g. per 100 cc.) 1 2 .0 6 .1 6 2 .2 4 1 2 .9 1 2 .2 1 1 .9 5 .9 3 6 .9 5 6 .9 1 1 .9 1 1 .9 4 2 .4 0 1 2 .5 1 3 .4 6 .2 5 6 .8 3 2 .6 3 3 .1 0 i 3 .1 1 2 .5 1 2 .5 6 .2 2 7 .7 6 6 .2 5 2 .3 8 2 .3 3 2 .5 8 1 1 .2 5 .0 0 4 .8 4 2 .1 3 2 .2 9 4 .2 2 3 .5 0 2 .8 1 1 .9 6 1 .6 1 2 ,2 9 8 8 .3 87 9 .0 9 .0 8 .0 8 9 .2 85 8 .6 9 .1 4 .4 7 7 .0 2 2 .3 6 2 .7 2 9 .9 9 0 .5 84 8.0 7 .5 4 .6 3 6 .2 5 5 .6 8 2 .5 3 2 .5 1 2 .6 0 9 1 .7 84 6 .4 7 .3 6 .4 8 4 .5 8 2 .8 2 2 .1 7 9 5 .0 85 8 .6 8 .6 4 .0 8 3 .8 1 2 .8 0 3 .0 2 7 6 .8 102 7 9 .0 98 83 5 98 9 4 .5 105 9 6 .5 102 1 2 .5 1 3 .0 1 2 .5 1 2 .1 1 2 .0 1 2 .1 1 1 .4 1 1 .1 1 0 .0 7 .6 8 .0 8 .5 1 1 .1 7 .4 9 .8 9 .6 8 .4 5 7 .9 1 6 .9 8 6 .9 8 6 .4 1 6 .3 2 6 .1 0 7 .0 0 7 .0 6 7 .7 9 6 .7 6 7 .1 8 1 0 .2 4 8 .2 3 7 .9 5 7 .0 2 2 .2 7 2 .7 5 2 .1 5 2 .6 5 2 .3 9 2 .9 6 2 .6 2 2 .3 7 2 .5 6 2 .1 6 2 .1 5 2 .2 4 2 .3 7 2 .5 1 2 .3 4 2 .9 7 (!) B orn M a y 3, 1932— fem ale. ( 2 ) A ge 275 d a y s. L igh t con v u lsio n s. (3) A ge 315 d a y s. Legs stiff. D ifficu lt to rise to feet. (4) B lo a t p robab le cause of d ea th . ( 6) B orn O ctober 14, 1933— m ale. ( 3) S lau ghtered . PATHOLOGY OF RICK ETS IN DAIRY CALVES Table 7. 29 Growth data and blood plasma analyses. Blood plasm a Age W eight H eight Calf N o. -7 W o (days) C -232(" )............................... (1) (2 ) (*) (<) Inorg. P Ca (lb.) (per cent normal) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 2 2 0 (2) 230 240 250 2 6 0 (3) 270 280 290 300 C) 310 319« 92 102 108 109 115 117 117 123 122 129 140 153 161 176 187 200 221 239 250 262 265 290 316 324 336 342 346 355 347 351 342 323 92 92 89 82 79 75 68 66 61 60 60 61 60 62 62 63 66 68 69 70 68 76 77 76 77 76 74 74 71 70 67 62 10 20 30 40 50 60 70 80 90 100 110 120 130 1400 150 160 161 (s) 100 109 120 134 148 158 162 174 183 201 229 231 245 259 276 275 100 98 99 101 102 101 95 94 92 93 98 93 92 91 91 87 B orn M a y 8, 1932— m ale. Stiff in front legs, K nees bow ed. Legs v ery stiff. Stiff in legs. B arely able to rise to feet. Mg (cm.) (per cent normal) 7 4 .8 100 1 4 .3 1 4 .3 6 .8 7 6 .6 9 2 .3 2 2 .1 9 7 8 .0 97 1 3 .9 1 3 .8 1 4 .8 5 .1 0 4 .5 5 7 .8 1 3 .4 3 2 .0 9 2 .5 3 8 1 .0 95 12.8 12.5 4 .4 6 4 .3 6 3 .0 2 1 .6 4 8 1 .8 91 1 2 .0 12.9 1 2 .8 8 4 .5 89 5 .8 7 5 .5 6 4 .4 3 5 .9 0 1 .5 4 2 .3 4 2 .0 5 2 .8 6 8 9 .3 89 9 3 .3 90 9 8 .5 92 10 1.5 93 1 0 4 .2 94 7 6 .3 104 7 9 .0 103 8 5 ,7 105 9 1 .3 103 9 4 .0 101 9 6 .3 98 (mg. per 100 cc.) 10.3 6 .0 4 1.76 9 .9 9 .3 10.2 6 .3 2 5 .8 4 5 .5 3 1 .7 8 2 .2 8 1 .8 0 11 .3 1 1 .2 6 .0 7 5 .6 3 2 .7 2 2 .7 4 1 0 .8 1 0 .4 10.1 6.01 4 60 5 .8 1 2 .8 8 2 .1 0 2 .4 5 9 .7 1 0 .9 9 .0 9 .0 5 33 4 .2 8 5 .1 7 3 .9 0 2 33 3 .4 7 2 .4 3 2 .9 3 1 3 .3 1 2 .8 13.1 12.9 1 1 .8 1 2 .0 9 .9 1 0 .0 9 .4 7 .7 7 .7 8 .0 8 .5 8 .0 8 .0 7 .9 6 .0 8 .0 1 8 .7 4 8 .5 6 8 .3 9 8 .8 7 7 .9 4 7 .2 3 7 .4 9 7 .1 9 6 .9 1 6 .1 8 6 .6 5 5 .9 9 5 .6 3 5 .7 9 4 .9 6 4 .9 8 2 .5 8 2 .9 0 2 .6 0 2 .4 6 2 .6 3 3 .2 5 2 .1 6 2 .0 7 1 .8 8 2 .9 7 2 .7 1 3 .1 6 2 .7 7 2 .5 7 2 .2 5 2 .5 4 2 .5 3 (6) Slaughtered. (') Born October 28, 1933— m ale. 0 ) L egs bowed. (8) Slaughtered. Slight p ittin g of articular surface on ball of femur. 30 M IC H IG A N T E C H N IC A L B U L L E T IN NO. 150 Table 8. Growth data and blood plasma' analyses. B lood p lasm a A ge C alf N o. (lb.) (per cent norm al) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 3 1 8 (2) 100 110 114 132 147 159 176 193 209 240 244 266 271 292 310 329 342 352 380 409 422 439 461 468 479 505 516 535 537 553 555 565 100 99 94 99 101 101 103 104 105 111 105 107 102 103 103 104 103 101 105 109 108 109 112 110 109 112 111 112 110 108 109 109 10 20 30 40 50 60 70 80 0 90 100 110 120 130 140(6) 1 5 0 (6) 1510 102 122 134 142 155 165 181 186 190 197 202 214 235 244 238 102 no 111 107 107 105 106 100 95 91 87 86 88 86 79 (days) C -237 (3) ................................ (0 (2) (3) (<) Inorg. P Ca B orn M a y 28, 1932— m ale. Slaughtered. B orn N ovem b er 10, 1933— m ale. C on v u lsio n at 82 d a y s of age and very irritable a t 83 days of age. (cm.) (per cent norm al) 7 7 .5 101 8 4 .3 103 8 8 .8 102 9 3 .2 101 Mg (m g. per 100 cc.) 1 4 .0 7 .4 9 2 .7 4 1 4 .0 1 5 .0 7 .2 3 7 .4 9 2 .3 8 3 .7 1 1 4 .4 1 3 .3 1 2 .6 1 2 .7 1 3 .0 6 .7 9 8 .0 1 7 .5 8 5 .9 5 7 .6 2 1 .9 4 2 .1 6 2 .3 3 2 .3 6 2 .2 2 1 1 .4 6 .9 1 7 .6 2 2 .7 2 1 .9 8 1 1 .3 1 1 .5 1 2 .2 7 .2 7 7 .6 2 7 .3 5 1 .6 1 2 .6 9 2 .5 9 1 2 .1 1 2 .7 8 .0 6 7 .0 6 3 .1 0 3 .0 2 9 7 .5 100 1 0 2 .2 100 1 0 5 .5 100 1 0 9 .5 102 1 1 .5 9 .8 1 1 .0 7 .8 1 8 .7 4 7 .8 1 2 .8 8 2 .7 5 2 .6 0 1 1 4 .5 104 10 2 9 .6 8 .7 4 8 .2 8 2 .5 5 3 .3 1 9 .5 8 8 9 .4 9 .1 3 9 .7 7 8 .5 0 2 .9 1 3 .2 4 3 .3 1 1 2 .2 12 4 1 3 .0 1 3 .5 1 1 .5 1 1 .7 1 0 .1 6 .4 8 5 7 2 8 .8 8 8 6 .7 6 .5 8 .5 6 6 .3 8 6 .8 3 6 .5 8 6 .6 9 6 .2 1 7 .6 2 7 .6 6 6 76 7 .6 7 8 .0 6 8 68 8 06 7 .4 5 2 .2 5 2 80 2 .7 2 2 .3 3 2 .2 0 2 .0 7 1 .7 0 1 .9 9 1 .7 5 1 .6 5 2 .4 5 2 .2 7 1 .83 1 .8 4 7 6 .7 102 8 1 .3 101 8 9 .8 103 9 0 .3 98 9 4 .7 98 (5) C onvulsion s at 139, 140 and 143 d a y s of age. CO C onvulsion . U n ab le to sta n d on rear legs. (7) Slaughtered. F ractu red verteb ra. PATH O LOG Y OF RICK ETS IN DAIRY CALVES T a b l e 9. 31 G r o w t h d a t a a n d b lo o d p la s m a a n a ly s e s . Blood plasm a A ge W eight H eight Calf N o. Ca (days) C-238C1) ............................... (lb.) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 161 P) (>) B o m N ovem ber 10, 1933— male. (2) Slaughtered. 113 109 122 130 145 160 184 205 229 253 269 290 314 336 353 370 (per cent normal) 113 98 101 98 100 102 107 110 115 117 116 116 118 118 117 116 (cm.) (per cent normal) 78 0 104 8 4 .0 105 9 3 .2 107 9 6 .7 105 1 0 3.3 107 Mg Inorg. P (mg. per 100 cc.) 1 3 .3 12.6 1 2 .5 1 2 .0 1 2 .7 11 .6 9 .3 9 ,5 8 .5 8 .7 8 .6 8 .2 8 .1 8 .0 8 .4 7 7 .6 2 6 .9 2 6 .1 3 6 .8 9 7 .4 9 6 .9 0 7 .0 2 6 .7 9 7 .1 6 6 .3 5 5 .6 4 6 .2 2 5 .9 2 2 .6 1 2 .4 7 2 .4 5 2 .3 7 2 .2 2 3 .1 1 2 .1 7 2 .1 2 2 .2 0 2 .1 6 2 .2 4 2 .2 3 2 .4 7 2 .4 7 32 MICHIGAN T E C H N IC A L B U L L E T IN NO. 150 F i g . 1. C a lf C -148. S e v e r e r ic k e t s . S h o w s e m a c ia t io n , h u m p i n g o f b a c k , s w e l li n g o f j o in t s , k n u c k li n g o f p a s t e r n s , a n d b o w in g o f l e g s . T h is c a lf w a s u n a b le to w a l k a n d w a s o n l y a b le t o s t a n d lo n g e n o u g h to b e p h o t o g r a p h e d . F i g . 2. C a lf C -238. T h is c a lf r e c e iv e d a b a s a l r ic k e t o g e n ic w h o le m ilk . S h o w s a b s e n c e o f d e f in i t e c lin ic a l s y m p t o m s s e v e r e r ic k e t ic c h a n g e s in t h e r ib a s s h o w n in F i g s . 12, a n a l y s e s in T a b le 9. T h is c a s e i s a g o o d ill u s t r a t io n o f w h ic h m a y b e p r o d u c e d m t h e r ib b e f o r e t h e l e g b o n e s s h o w r a tio n s u p p l e m e n t e d w i t h n a t u r a l o f r ic k e t s . N o te , h o w ev er th e 20, a n d 26. N o t e ’a l s o t h e b lo o d t h e e x t e n s i v e r ic k e t ic a l t e r a t i o n s c li n i c a l e v id e n c e o f t h e d i s e a s e . PATHOLOGY OP PICKETS IX DAIPV ( ALYF.S F i g . 3. C a lf C -231. N o r m a l. S h o w s : r e s t in g c a r t ila g e , A ; c e ll p r o life r a tio n , B ; c e ll g r o w t h , C ; m a tu r e c a r t i la g e c e lls w it h c a lc ifie d m a tr ix b e t w e e n t h e r o w s of c e lls , D ; v a s c u la r b u n d le , E . ( T h is a n d a ll s u c c e e d in g p h o t o m ic r o g r a p h s illu s t r a t e m id - f r o n ta l s e c t io n s t h r o u g h t h e le f t e ig h t h c o s to c h o n d r a l ju n c tio n . T h e d ia p h y s is is to t h e r e a d e r ’s r ig h t, e x c e p t in F i g . 14 w h e r e i t is t o th e b o t to m o f t h e p a g e ) . 120X. F ig 4 A r e a A in F i g . 3. S h o w s : m a tu r e c a r tila g e c e ll, A ; is o g e n o u s p a ir of im m a tu r e c a r t ila g e c e lls , B; c a r tila g e m a tr ix , C. 460X. 34 M ICHIGAN T E C H N IC A L B U L L E T IN NO. 150 F i g . 5. C a lf C -233. N o r m a l. S h o w s : o s t e o c l a s t s , A ; c o r e o f c a lc i f i e d c a r t i l a g e m a t r i x , B ; d e p o s itio n o f g r a n u l e s o f in o r g a n ic s a l t s in s t r i a t i o n s , C . 4 6 0 X . F i g . 6. F r o m n o r m a l s p e c im e n . S h o w s : o s t e o c l a s t s , A ; p e r io s t e u m , B . N o t e l a r g e n u m b e r s o f o s t e o c l a s t s h e r e in t h e r e g io n w h e r e t h e t r a b e c u l a e m e e t t h e p e r io s t e u m . 1 2 0 X . PATHOLOGY OF RICKETS IN DAIRY CALVES F i g . 7. C a lf C -231 . N o r m a l a d v a n c e m e n t of e m b r y o n ic m a r r o w . S h o w s : m a tu r e c a r t ila g e c e lls , A ; c a lc ifie d m a tr ix b e t w e e n m a tu r e a n d d e g e n e r a tin g c a r t ila g e c e lls , B ; c o a g u la t e d b lo o d e le m e n t s in s m a ll s a c -lik e a r e a s in z o n e of c a r t ila g e r e m o v a l, C; t o n g u e o f e m b r y o n ic m a r r o w , D ; t r a b e c u la e of c a lc ifie d t is s u e , E ; o s t e o c l a s t s , F ; o s t e o b l a s t s , G . 120X . F ig 8 C a lf C -231. N o r m a l. S h o w s : v a s c u la r b u n d le s in r e s t in g c a r tila g e , A ; a r e a o f p r o lif e r a t io n a n d c e ll r o w s , B ; c a lc ific a tio n a t j u n c tio n o f c a r tila g e w ith th e d ia p h y s is , C ; t r a b e c u la e o f c a lc ifie d s u b s t a n c e D ; m a r r o w , E . N o t e th e r e g u la r a n d e v e n m a n n e r in w h ic h t h e d ia p h y s is jo in s w it h th e c a r tila g e n o u s e n d of th e b o n e . 30A. 36 M ICHIGAN T E C H N IC A L B U L L E T IN NO. 150 F i g . 9. C a lf C -175. M ild r ic k e t s . S h o w s : r e ta r d e d a n d ir r e g u l a r p r o v i s i o n a l c a lc i f i c a t i o n o f c a r t i la g e m a t r i x , A ; h e a v i e r d e p o s it s o f g r a n u l e s o f in o r g a n ic s a l t s in p e r ip h e r y o f l o n g it u d i n a l b a r of c a r t i la g e m a t r i x , B ; c o a g u l a t e d b lo o d e l e m e n t s in s a c - l ik e s t r u c t u r e , C ; o s t e o c l a s t , D ; o s t e o b l a s t , E ; e m b r y o n ic m a r r o w , F . 46 0 X . C alf C -170. M ild r ic k e t s . S h o w s : e x t e n s i o n o f t o n g u e s o f c a r ' v e r t e b r a l ly in to t h e d i a p h y s is , A . 3 0 X . PATHOLOGY OF RICKETS IN DAIRY CALVES F i g . 12. F i g . 11. C a lf C -233. N o r m a l. S h o w s : r e s t i n g c a r tila g e , A ; a r e a o f p r o life r a tio n and c a r t i ­ la g e c e ll r o w s , B ; ju n c tio n o f t h e d ia p h y s is w it h th e c a r tila g e n o u s en d o f th e r ib — th e s li g h t s e p a r a tio n n o t e d h e r e is an a r tif a c t , C ; b a r s o f b o n e t r a b e c u la e — n o te t h e ir d a r k s ta in , h o w th e y e x t e n d in a d ir e c tio n p a r a lle l to th e lo n g a x is o f th e b o n e , a n d h o w t h e y d e c r e a s e in n u m b e r a n d b e c o m e h e a v ie r f u r th e r in th e d ia p h y s is , D ; m a r r o w , E ; t h ic k e n in g o f p e r i­ c h o n d r iu m , F . 5X . F i g . 12. C a lf C-238. S e v e r e r ic k e t s . S h o w s : r e s t i n g c a r t ila g e , A ; a rea o f p r o life r a tio n a n d c e ll r o w s , B ; in c r e a s e d d e p th o f z o n e o f m a tu r e c a r t ila g e c e lls , B 1; a r e a of c a r tila g e r e m o v a l— n o t e i r r e g u la r it y in t h is a r e a , C ; s h o r t, s tu b b y t r a b e c u la e , D ; s a c - lik e s t r u c t u r e s iso la te d in ir r e g u la r ly - t h in n e d c a r t i la g e , E : e m b r y o n ic m a r r o w a n d o s te o id t is s u e , F . N o t e ir r e g u la r a d v a n c e m e n t of th e m a r r o w a n d its e x te n s io n v e u t r a lly in th e a x ia l r e g io n o f t h e b o n e . SX. 37 33 M ICH IGA N T E C H N IC A L B U L L E T IN NO. 150 c* * . F i g . 13. C a lf C -233. N o r m a l. S h ow s: o s t e o c l a s t , A ; o s t e o b l a s t b o r d e r in g a t r a b e c u l a o f b o n e — n o t e e c c e n t r ic a l ly p la c e d n u c le i, B ; o s t e o b l a s t s u r r o u n d e d b y o s t e o i d t i s s u e a n d a l s o p a r t ly s u r r o u n d e d b y d a r k g r a n u l e s o f in o r g a n ic s a l t s — s u c h c e ll s a r e g r a d u a l ly c h a n g i n g i n t o o s t e o c y t e s , C ; o s t e o i d t i s s u e , D : c o r e o f c a lc i f i e d c a r t i l a g e m a t r i x a s y e t u n r e m o v e d b y c h o n d r o l y s is , E . 460X . l ' i g . 14. C a lf C -151. N o r m a l. S h o w s : g r o u p s o f o s t e o b l a s t s a s t h e y o c c u r o n t h e b o r d e r s o f t h e la r g e r t r a b e c u la e m t h e v e n t r a l e n d o f t h e d i a p h y s is , A . 46QX. PATHOLOGY OF RICKETS IN DAIRY CALVES 39 F ig 1. 15. C a lf C -233. N o r m a l. S h o w s : e m b r y o n ic m a r r o w , A ; t r a b e c u la e o f b o n e , B ; o s t e o ­ b la s t s b o r d e r in g tr a b e c u la e , C ; o s t e o c l a s t s o n e n d s o f t r a b e c u la e w h e r e t h e y a r e a s s o c ia t e d w it h t e r m in a l e r o s io n o f th e c a lc ifie d t r a b e c u la e , D . 120X. F i g . 16. A r e a D in F i g . 15. S h ow s: te r m in a l e r o sio n b y o s t e o c la s t s , D . 575X . MTCTTTCAN T E C H N IC A L B U L L E T IN NO. ISO 40 . t j .*» "* ” .A , E - *, *■ i * / 4 , 1 » * • ' ,<1 / 0 ' 1 » / f 1 ' , F i g . 17. , ■ 4 : " ,ri • / ✓ / ♦ ■\ < 1 ' • i , / -I) . ■ . i V- . •,? * *■- / ’ •' W W ‘ ' , // V *• 1 D < * v ' f v • • . . '' ' *• ' yT ^ ‘- V ’ O 1 •. ^ '.'/■< " > ■ < , * ^ > , *l < / , V » • ~s ' , > ♦ v \ -J ' ' v" f , , • ' ■• . o , . • 1 ' * • ■ ( * —' <1' \ , ; ; • > a I s C % %I -A' ’|S . L * ’ m 1 Cm . ' 1. ' ' ' . , ■ ‘’ 1 * l> . C a lf C -15S, S e v e r e r ic k e t s . S h o w s : r e s t i n g c a r t i la g e , A ; c e ll p r o lif e r a t io n , B ; c e ll g r o w t h , C ; e m b r y o n ic m a r r o w , D ; w a l l o f b lo o d v e s s e l , E . 1 2 0 X . m ( j F i g 18. A r e a A in F i g . 17. S h o w s : i n c r e a s e d n u m b e r o f i s o g e n o u s p a ir s o f c a r t i l a g e c e l l s in s h r u n k e .^ o n d .t i o n , B . N o te d e c r e a se m a m o u n t of m a tr ix a n d c o m p a r a tiv e a b se n c e o f m a tu r e PATHOLOGY OF RICKFTS IX DAIRY CALVES 41 P i g . 19. C a lf C -237. A d v a n c e d r i c k e t s . S h o w s: l a r g e n u m b e r of s a c - l ik e s t r u c t u r e s , A ; l e n g t h e n e d r o w s of m a t u r e c a r t i l a g e c e lls , B ; i r r e g u l a r l y s h a p e d t r a b e c u l a e of c a lc if ie d t i s s u e , C ; a r t i f a c t , D . 30X . F i e 20 C a lf C -238 S e v e r e r i c k e t s . S h o w s : s a c - l ik e s t r u c t u r e s , A ; e n o r m o u s ly in c r e a s e d d e p t h o f z o n e o f m a t u r e c a r t i l a g e c e lls , B ; i r r e g u l a r l y s h a p e d t r a b e c u l a e of b o n e C ; e m b r y o n ic m a r r o w a n d o s te o i d t i s s u e D . N o t e i r r e g u a r a d v a n c e m e n t , v e n t r a l l y , o f t h e e m b r y o n i c m a r r o w F t h e a x F l r e g i o n o f t h e b o n e . C o m p a r e w i t h F i g . 19, C a lf C -237, w h ic h h a d r i c k e t s o f s im il a r s e v e r i t y . ' a s i n d i c a t e d b y t h e b lo o d p i c t u r e f o r a b o u t t h e s a m e l e n g t h o f t im e b u t m a d e s u b ­ n o r m a l g a i n s in b o d y w e i g h t a n d d e v e lo p e d m u c h le s s s e v e r e b o n e le s io n s . 30X . 42 M ICHIGAN T E C H N IC A L B U L L E T IN NO. 150 F i g . 21. C a l f C -171. S e v e r e r i c k e t s . S h o w s : s p l i t t i n g o f c a r t i l a g e , A ; o s t e o i d a n d f i b r o u s c o n ­ n e c t i v e t i s s u e s , B ; c r u s h i n g o f c a r t i l a g e c e ll s , C ( s e e F i g . 22 f o r h i g h e r m a g n i f i c a t i o n ) ; p a t c h e s o f o s te o i d t i s s u e a b o u t i n d i v i d u a l c e ll s , D ( s e e F i g . 24 f o r h i g h e r m a g n i f i c a t i o n ) , 3 0 X . F i g . 22. A rea C in F i g . 21. S h o w s : c r u s h i n g o f c a r t i l a g e c e ll s r e j u v e n a te d c a r t i l a g e c e lls , B . 460X . A- PATHOLOGY OF RICKETS IX DAIRY CALVES F i g . 23. C a lf. C -171. S e v e re ric k e ts . S h o w s : e m b r y o n i c m a r r o w , A ; o s te o i d a n d f ib r o u s c o n n e c t i v e t i s s u e , B ( r e f e r to B i n F i g . 2 1 ); p a t c h e s o f o s te o i d t i s s u e s u r r o u n d i n g i n d iv i d u a l c e lls , C ; e a r l y o s s if ic a t io n in p a t c h e s of o s te o i d t i s s u e s i m i l a r to t h o s e a t C , D , ( n o te c h a r a c t e r i s t i c h a lo e f f e c t d e s c r i b e d in t e x t ) ; r e o r g a n i z a t i o n o f t h e lo o se c o n ­ n e c t i v e t i s s u e b e tw e e n t r a b e c u l a e g i v i n g r is e to e m b r y o n i c b o n e m a r r o w , E ; t r a b e c u l a w i t h c o r e o f b o n e a n d m a r g i n o f o s te o i d t i s s u e b o r d e r e d b y o s t e o b l a s t s , F . 120X. F i g . 24. A r e a D i n F i g . 21. S h o w s : i n d iv i d u a l c e lls s u r r o u n d e d b y c i r c u l a r z o n e s o f o s te o i d t i s s u e w h ic h s t a i n d e e p ly a c id o p h ilic , A . 460X . MICHIGAN T E C H N IC A L B U L L E T IN NO. 150 C 188 C 238 C 171 C 231 F i g . 25. R o e n t g e n o g r a m s ‘if m id ■f r o n t a l s e c t i o n s 4 m m . in t h i c k n e s s t a k e n f r o m t h e left, e i g h t h c o s t o c h o n d r a l j u n c t i o n o f e a c h c a lf a s l a b e le d . T h e v a r y i n g s h a d e s o f b l a c k s h o w t h e d i s t r i b u t i o n of c a lc i f i e d t i s s u e s . ( -151 s h o w s a n o r m a l c o n d i t i o n . C -1 4 8 , C -155, a n d C -171 s h o w r i c k c t i c c h a n g e s a s e v id e n c e d b y d e f i c ie n t c a l c i f i c a t i o n a t t h e e n d s o f t h e b o n e s . T h e r o e n t g e n o g r a m of ('-1 S 8 s h o w s s l i g h t e v id e n c e o f a t l e a s t t w o n r r e s t e d - g r o w t h l i n e s i n t h e v e n t r a l e n d o f t h e d i a p h y s i s . X o t e t h a t b e a d i n g of t h e r i c k c t i c r i b s is m o r e p r o n o u n c e d o n t h e m e d i a l s id e . A l s o , n o t e i n c r e a s e d c u r v a t u r e o f r i c k e t i c r i b s in a m e d i a l d i r e c t i o n . F i g . 2(>. R o e n t g e n o g r a m s o f m i d - f r o n t a l s e c t i o n s 4 m m . in t h i c k n e s s t a k e n f r o m t h e l e f t e i g h t h c o s t o c h o n d r a l j u n c t i o n o f e a c h c a lf a s l a b e l e d , f 1-224 s h o w s e v i d e n c e o f e p i p h y s e a l t y p e of h e a l i n g r i c k e t s ( r e f e r t o T a b l e 6 ) . C -2 2 9 , ('-2 3 1 , a m i ('-2.33 a r e f r o m n o r m a l c a l v e s . C -2 3 2 , C -237. a n d ( -258 a r e f ro m r i c k c t i c c a l v e s . X o t e t h a t t h e r i c k e t i c a l t e r a t i o n s i n C -2 3 7 a r e r e l a t i v e l y m ild w h e n c o m p a r e d w i t h (1-258, b e c a u s e of p o o r g r o w t h ( r e f e r to T a b l e s 8 a n d 9, a n d F i g s . ]9 a n d 20). PATHOLOGY OF RICKETS IX DAIRY CALVES F i g . 27. M i d - f r o n t a l s e c t i o n s 4 m m , in t h i c k n e s s t a k e n f r o m t h e le f t e i g h t h c o s t o c h o n d r a l j u n c t i o n of e a c h c a lf a s la b e le d , a n d s ta i n e d in s il v e r n i t r a t e s o lu t io n . S h o w s t y p i c a l a p p e a r a n c e o f n o r m a l a n d of r ic k e ti c r ib s w h e n t r e a t e d in t h i s m a n n e r . C o m p a r e w i t h F i g . 26 . N o t e t h a t w h ile t h e g e n e r a l a p p e a r ­ a n c e s a r e t h e s a m e , t h e r e is g r e a t e r d e ta i l in t h e r o e n t g e n o g r a m s t h a n in s p e c i m e n s s t a i n e d in s i l v e r n i t r a t e s o lu tio n . F i u 28 R o e n t g e n o g r a m s o f m id - f r o n t a l s e c t i o n s 4 m m i n t h i c k n e s s t a k e n f ro m t h e l e f t e i g h t h c o s to c h o n d r a l j u n c t i o n of e a c h c a lf a s la b e le d . C -176 s h o w s s o m e r a r e f a c t i o n ; t h i s c a lf w a s f r e e f r o m r i c k e t s . C -170, C -173, a n d C - 17a a r e s p e c im e n s f ro m r i c k e t i c r ib s I n c a s e C h l7 5 , d e f i n i t e e v id e n c e o f r i c k e t s w a s i n d i c a t e d o n ly in t h e b lo o d a n a l y s e s a n d in h i s t o l o g i c a l s t u d i e s o f t h e b o n e . 45 46 M ICHIGAN T E C H N IC A L B U L L E T IN NO. 150 F i g . 29, C a lf C -232. S e v e r e r i c k e t s . S h o w s c h a n g e s s i m i l a r t o t h o s e in F i g . 20. C o m p a r e w i t h F i g . 10, C a lf C -1 7 0 , s h o w i n g c o m p a r a t i v e l y s l i g h t a l t e r a t i o n s . F i g . 29 is f r o m a y o u n g e r c a lf w h ic h h a d r i c k e t s f o r a m u c h s h o r t e r t im e t h a n d id C -1 7 0 . T h i s i l l u s t r a t e s t h e m o r e e x t e n ­ s iv e a lt e r a t io n s w h ic h a r e p ro d u c e d in r ic k e ts in y o u n g e r c a lv e s . 30X . F i g . 30. C a lf C -1 8 8 . A d v a n c e d r i c k e t s in a n o l d e r c a lf . S h o w s : c a lc ific a tio n of m a tr ix , A ; e m b ry o n ic m a r r o w a n d c o n n e c tiv e tis s u e , B ; m o re th a n u s u a l n u m b e r of b o n e t r a b e c u l a e , w h ic h a c c o u n t f o r t h e a r r e s t e d - g r o w t h l i n e s i n d i c a t e d in F i g ?5 C 30X PATHOLOGY OF RICKETS IX DAIRY CALVES F i g . 31. C a lf C-14S. R ib s e p ip h y s e a l e n d s of th e b e st seen a t A . f r o m t h e r i g h t s id e . S h o w e n l a r g e m e n t s o f t h e v e n t r a l r ib s . T h i s c o n d itio n is c o m m o n ly r e f e r r e d to a s b e a d in g , a n d is F i g . 32. C a lf C -1S8. R o e n t g e n o g r a m s o f t h i n s p e c i m e n s o f b o n e ta k e n th ro u g h th e p r o x im a l e n d of t h e f e m u r . T h e r e a r e n o a b n o r m a l i t i e s a t A . b u t n o t e t h e m o d e r a te r a r e f a c t i o n a t B a n d t h e lo s s o f c a r t i l a g e a n d r a r e f a c t i o n a t C. S e e F ig . 33. F i g . 33. C a lf C -188. P r o x i m a l e n d o f f e m u r o f r ic k e ti c c a lf . S h o w s : p i t t i n g o f b o n e , A ; d e s t r u c t i o n o f c a r t i l a g e , B ; t h i n n i n g of c a r t i l a g e , C . ( N o t e t h a t t h is s p e c im e n h a s b e e n r e c o n s t r u c t e d t h r o u g h t h e c e n t e r a t D w h e r e a s e c tio n o f b o n e w a s s a w e d o u t f o r h i s t o lo g i c a l s t u d y ) . E x t r e m e p i t t i n g a n d e r o s i o n o f t h e a r t i c u l a r s u r f a c e s , s u c h a s i l l u s ­ t r a t e d h e r e , o c c u r r e d b u t o n c e in c a lv e s u s e d in t h is s tu d y . F i g . 34. H u m e r u s o f a r ic k e ti c c a lf . S h o w s : a c o m m o n c o n d it i o n in r i c k c t i c c a lv e s . p i t t i n g of a r t i c u l a r s u r f a c e A . T h is w as