. . — \v .‘o . a v . . . .\ .-‘\a _. .\\. .95 . . u w» o. .. n. ...m .7 .m r. .5 2 a . o I: 6‘! u . P. JV . .0.” ~ g. o u _ a. ., ... u . ' ‘ c" /.\. . . 3.x” “ _. hp. w... J H , .. n . n o . o s, . , . c... . V r; I ~ a“ u. n E. . . 7 . . .... . .. . . .. . ... .u g ”a. 7 .J m;- c. r...\ . . ... é ... . \i . . fl. ,. .. . .. .2 a v S C a . I” O o; c. ‘ .. \ . a‘ J“ '1 \I n . M». R. 1 . 9.. .3 ... . . a 1. . :3... g r g . . . ..‘...V 2 2 a m.“ . Poo _. a ,1. . x S 4 . .t X. . 7.. v .2; c.\.( .‘ . . .N $5.. . . r a .1.\ . \\ .‘ to. H . 53 a r K « 9:. v .0 .5. . , . . . r s. a. . c.. a .... o -. rr 9 a 4 . .. .4 \y. I § 0.. .\ro¢ tldv - O O I. 1 : \BV u‘a . 2 “mm...“ a o .\ I. .u.. . . 4x . It I; .u.. ". fl, 8 in t“ '5: ... 'd. ‘8 u -—-- V'—-- r i i E lg?“ ’ ‘_ '. ',)1I:r .y’ “A V.» -' .. ' .s‘ I ’ )0 1 :- 1, . This is to certify that the thesis entitled Ionic and Bound Calcium Levels in the Blood of Cows Suffering from Partnrient Paresis (Milk Fever) presented by Edward A. White has been accepted towards fulfillment M. 8. of the requirements for degree 111M Maj I. . fessor ._.-\ IONIC AND BOUND CALCIUM LEVELS IN THE BLOOD OF CONS SUFFERING-FHOM PARTunIENT PAhESIS (MILK FEVER By Edward Alexander white A THESIS submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in.partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy Husbandry 1952 ACKNOWLEDGMLNTS The author expresses his appreciation to Doctor C. F. Huffman, Research Professor of Dairy Husbandry, for his council In connection with the work involved in this study. Appreciation is also extended to Mr. R. M. Grimes, Instructor in Agricultural Chemistry, who gave generously of his time in making the necessary blood analysis for pertinent data in this study. A word of thanks is given to William Rakestraw, a senior veterinary student, who provided the photographic plate of the equipment used in this study. Work of a kind necessary to complete a manuscript con- tributory to a clear and perhaps more complete understanding of a subject requires sacrifices that are not alone shared by the author but by his wife and family as well. To them this statement of appreciation is made for their steadfast- ness. To them this work is dedicated. %%*%%*%%%%% J,_.::_ ‘7 _v_£.p' _\I_V_ A \l\ AAA V \I_J.r__‘n_v__\a ‘L K AA A h n‘k iii-31% x 33%? 3 fir. TABLE OF CONT LL‘NTS INTRODUCTION 00.0.0...O0.0.0...OOOOOOOOOOOOOOOOOOO00.0.0...0...... 1 RHJIIM OF LI’l‘niuTUlufl...................... ..... .................. 2 Introduction. ...... ......................................... Early TheoriGS.............................................. Cerebral Anemia Theory...................................... Cerebral Congestion.Theory.................................. Ap0plexy Theory............................................. bacterial Infection of Uterine Origin Theory................ Bacterial Infection of Mammary Origin Theory.........,....., Anaphylaxis Theory.......................................... Alite-intoxication Theory.................................... Hypoglycemia Theory......................................... Hypocalcemia Theory......................................... 1h Parathyroid Deficiency Theory..... ..... ................ 21 Vitamin D Administration as Preventative............... 2h Magnesium hareosis Theory................................... 25 Summary of the Review of Literature......................... 25 }_.J Ommwwopwwm OBJECT OF THIS STUDY...... ...... ................................. 28 ImmmnmmmLPMEEMML.H.H.H.H.H.”.H.H.H.u.u.n.u.u 29 RESULTS.......................................................... 3h DISCUSSION.................... ........ ........................... 39 RfimNC/Esooooooococo.oooooooooooooQOoooooooooQOooooooooooooooooo Ml INTRODUCTION Parturient paresis (Milk Fever) ranks high in economic importance to the dairy industry when consideration is given solely to such diseases as are classified under the heading of metabolic diseases in the bovine. It has been the general observation of veterinarians and research workers, both in.Eur0pe and in.America, that the incidence of parturient paresis has increased along with the increased productivity of the dairy cow. This primary acknowledgment based on statistics compiled at various veterinary clinics has been the basis for studies relating to the etio- lOgical factor or factors in this disease. Concurrent with this observation statistics have also indicated that the incidence of the disease increases up to the age at which the dairy cow attains the high- est level of production and then decreases as the productivity declines with advanced age. The true underlying cause of the occurrence of this disturbance has not been revealed.. The work of investigators has, however, lead to the discovery of a satisfactory therapeutic procedure that can be relied upon to correct the disturbance. It is with the intent to supplement, and possibly alter in some cases, part of the prevailing data relative to the phase of study that has been undertaken. REVIEW OF LIT EMITU RE Introduction Hutyra and Marek (1922) defined parturient paresis (milk fever) thus: "An afebrile disease which occurs commonly at the termination of parturition and which is manifested by a sudden loss of conciousness and a generalized paretic condition." Law (l923-2h) defined the disease as a nervous disorder which develOps in plethoric cows, heavy milkers after calving, and was char- acterized by loss of sense, of conciousness, and of muscular control: by hypothermia, convulsions, coma, and mellituria. The disease parturient paresis was first described by German veteri- nary surgeons at the beginning of the nineteenth century. The time at which the disease first manifested itself corresponded to that period of time in history when dairymen became interested in increasing milk pro- duction through generous feeding. As the years passed the efforts on the part of dairymen throughout the world have, for competitive and economic reasons, been intensified to increase the individual production of all cows of all breeds. A better understanding of genetics, nutrition, and animal husbandry has made possible the progressively higher level of production of dairy cattle of all breeds everywhere. In order to make known the thinking of the eighteenth century veterinarian with respect to this disturbance Ralph (1850) stated that the incidence increased with years and that heavy milkers, after a few calvings, were more liable to this disturbance. Early Theories Over the many years that this disease has been investigated several theories have been advanced, many of which time and subsequent research have proven unfounded. In order to provide basis for further study certain of the older theories should be given recognition irre- spective of the fact that experimental evidence has, at least in part, failed to support them. Prominent among these theories are the follow- ing: cerebral anemia, cerebral congestion, apoplexy, bacterial infection of uterine origin, bacterial infection of mammary origin, anaphylaxis, auto-intoxication, hypoglycemia, magnesium narcosis, and hypocalcemia. Investigators, as the review of literature bears out, have given serious consideration to certain physiological processes and their progressive adjustments, as gestation progressed, to meet the changing exigencies concurrent with gestation and the ultimate parturition. A lack of a complete understanding of all of the physiological transi- tions attendant in the female during gestation and parturition compelled them to supplement known facts with theory. Cerebral Anemia Theory The theory of cerebral anemia was one of the first to be considered by such workers as Glass (lSBh), Billings (lfibh), Frank (1871), Thomassen (1890), McConnell (1890), and more recently by Spiegel (1939). A review of the work of the above men revealed the following fact as the basis for the belief in this theory. Immediately previous to and at the time of parturition the mammary system undergoes hypertrOphy with accentuation of the blood vascular system in preparation for lactation. This was the basis for the theory that the sudden increased demands of the mammary system for blood simultaneously caused a sudden blood deprivation of the brain. The final result was cerebral anemia and a semi-comatose to comatose condition that could, unless checked, lead to death. As late as 1939 this theory had strong adherents and it was believed that the then effective therapeutic procedure of air insufflation of the udder was responsible for the relief of cerebral anemia. The work of Hutyra and Marek (1926) discredited this theory since they were unable to produce the parturient paresis syndrome by removing nearly 50 per cent of the blood from a cow. Cerebral Congestion Theory In almost direct contradiction to the cerebral anemia theory was that of cerebral congestion (hyperemia) supported by such investigators as Noquet (1853), Sanson (léSh), Ayrault (1857), Gerrard (1879), and Cox (188h). A commonly practiced therapeutic procedure followed shortly before and at this time was that of bleeding as described by Youatt (1830). In order to emphasize the degree of reliance placed upon bleed- ing as a therapeutic as well as a prephylactic procedure, Youatt (1830), who was a practitioner of veterinary medicine in good repute is Quoted, "If the cow is in a high or dangerous state of condition and has been fed on luxuriant pasture, it will be very proper to bleed her and give her a dose of physio and remove her to a field of much shorter bite a few days before her expected time of calving. I believe that many valuable animals have been saved by this precaution." Stewart (1890), while admitting having been singularly unfortunate in the treatment of this disturbance considered the practice of abstracting large quantities of blood as being therapeutically correct. Rogerson (16h0), a con- temporary practitioner, reported on venesection for the withdrawal of large quantities, four quarts, of blood as an important part of the Ptherapeutic handling of parturient paresis cases. However, this manner of therapy, applied with the apparent intent to relieve circulatory congestion, was followed without warranted Success. During the years in which these contemporary workers were endeavoring to uncover the cause of this mysterious parturient condition a therapeutic procedure that could be depended upon for the relief_of the condition had not been found. ConseQuently a considerable number of subjects died and were presented for autOpsy. Foetal (1855) maintained that he had found several cases in which blood clots were found under the arachnoid cover- ing of the brain. Sanson (lSSh) attempted to account for hyperemia of the cerebrum by the fact that physiologically the womb, following parturition, was faced with a sudden departure of blood concurrent with uterine involution. This sudden dimunition of blood to the uterus threw an additional volume of blood upon the general circulatory system more particularly the brain. In conseguence of the hyperemia produced there a rupture of the capillaries, it was believed, sometimes occurred. The belief that blood clots in the brain were a common finding in this con- dition could not be credited for the reason that the presence of the clots would constitute a pathological state the repair of which, if at all, would be slow and totally inconsistent with the rapid rate of recovery of cases following rational treatment. Apoplexy Theory Concurrent with the theory of cerebral congestion was that of apoplexy which had as its supporters Thacker (ldhl), Festal (1855), Halley (.1872), Simonds (181:1), um (1892-93), and Gerrard (1579) . While the etiology of this state was believed to be associated with ex- cessive blood vascular tension or pressure in an indirect way, in that the increased pressure resulted in a disturbance of the spinal and cerebral nervous systems, the phenomenon was related as being primarily nervous in nature. Various physiological abnormalities or, better stated, adjustments that accompany gestation and parturition, it was believed, thrust upon the nervous system shocking disturbances accountable for apOplexy. Emboli, that could very probably have been introduced into the capillaries that supply the uterine caruncles at the time the placental attachments were broken, were believed, in some cases, to be responsible for thrombosis of the cerebral blood supply with resultant apOplexy. 'Winc0p (1868) reported in support of the theory of Simonds (lBhl) that the disease was centered in the brain and nervous system. Thus it can be recognized that early investigators were concerned with the role of faulty circulation, and of certain nervous anomalies and their probable association with parturient paresis. Bacterial Infection of Uterine Origin Theory It is not surprising that the interest of investigators turned to the uterus as a possible haven for some substance or possible contami~ nent that could be a contributing factor in parturient paresis. It was less surprising, too, that attention was directed to this organ since it is conspicuously active during and at the close of gestation. Prominent among the workers who studied the bacterial flora of the uterus with in- tent to determine its probable role in parturient paresis were Lanzilloti- Buonsanti (1890), Zundel (1890), Lyman (187:5), Sclunidt-Mulheim (1590) (19014), Friedberger and Frohner (189s), Nocard (1896-97), and Guillebeau (191.5) . It was generally believed by these investigators that bacteria of the anaerobic type, which could already be present in or introduced at the time of parturition into the uterus, could quite probably create toxins which when absorbed could have a depressant effect upon the central, as well as, the vasomotor nervous systems. Thus the uterine infection theory commanded the attention of the above mentioned investigators. Some of these same investigators, however, associated the mammary gland with the onset of parturient paresis. Bacterial Infection of Mammary Origin.Theory In the udder as in the uterus it was believed possible that certain anaerobic organisms comfortably situated within the gland and vegetating while feeding upon the colostrum could produce toxins that could, as in the uterus, have the effect of a depressant upon the central nervous system. It was this belief shared by Thomassen (1890), Knusel (1903), and Schmidt (1897) that led to the therapeutic procedure of injecting potassium iodide solution into the udder with intent to destroy the microbes and thus effect relief from the symptoms of central nervous toxemia. Since the anaerobes were the organisms believed by Kunsel (1903) to be reSponsible for the toxins it was reasoned that the intro- duction of oxygen into the udder would alter the environment in a way to inhibit the activity of the anaerobes. This belief in anaerobic involvement was abandoned, however, but the fact remained that the mortality rate was lowered following inflation of the udder. The in- flation of the udder with air became the standard therapeutic procedure in the treatment of cows affected with parturient paresis. Its applica- tion resulted in a marked decrease in the mortality rate in this malady. This surprising fact spurred research workers to seek an answer to the question as to why inflation of the udder with oxygen or sterile air could favorably affect the course of this disease. Anaphylaxis Theory About the same time that work with the uterus and the udder and the bacterial invasion thereof was being done certain workers advanced the theory of anaphylaxis. Prominent among these investigators were HutyradMarek (1926), and van Goidsenhoven (1927) who reasoned that there could be a protein sentitization following the absorption of milk- casein during the previous lactation. The sensitivity that develOped manifested itself in the succeeding lactations when further absorption of milk-casein could take place. This phenomenon was believed to be aided by alexin, a protein antibody substance normally present in the blood. This hypothesis could not be confirmed by the experimental in— jection of placental emulsions of milk and of colostrum immediately after parturition. Auto-intoxication Theory Menig (1925) theorized that toxic substances likened to ptomaines could be produced in the pregnant uterus which could, when absorbed, exert a depressant effect on the central nervous system and upon the vasomotor center as well. There were two contemporary preponents of the auto-intoxication theory, Menig (1925), and SchmidtéMihlheim (1890) (l90h). The work of these investigators was superseded by that of Schmidt-holding (19st). The latter attributed the auto-intoxication phenomona to the absorption from the udder of leucomames which were considered to be the end products from the decomposition of colostrum. Schmidt-Kolding reasoned that the treatment for this difficulty should be directed toward retarding secretion of the udder and at the sane time neutralizing the leucomames by the use of some autotoxic agent. He, therefore, advocated the injection of the udder with an aqueous solution of potassium iodide. The immediate decrease in the mortality rate following this therapeutic procedure gave the theory general recog- nition. This manner of therapy was tried with warranted success by many and in time certain modifications of this therapeutic procedure led to the discovery that the beneficial results obtained was largely, if not wholly, a result of the distention of the udder rather than the autotoxic action of the potassium iodide. To this able investigator 10 credit was given for the discovery of the inflation method of treatment which was used for many years with a high degree of success in correct- ing parturient paresis. Hypoglycemia Theory Following the advent of the udder inflation method for treating parturient paresis very little was done for a time to expand the general knowledge with respect to the basic cause of this malady. Neff (1923), reported that a condition of hypoglycemia could be in- duced by insulin injection. The condition thus produced so nearly re- sembled parturient paresis that one veterinarian, whose name remains unknown, was stimulated to try the injection of glucose into the blood stream in a case of parturient paresis. It was reported that he effected a cure. This experience led investigators Widmark and Carlens (1925) of Sweden,.Auger (1927) of France, and Maguire (1926) of England, to center attention on the hypoglycemia theory; Widmark and Carlens re- ported the experimental production of hypoglycemia by insulin injections and the description of the symptoms manifested resembled closely those that were commonly observed in parturient paresis including the char- acteristic attitude of the head turned to rest on the chest. Auger (1927) later duplicated the work of Hidmark and Carlens (1925). Thus it was believed for a short time that a significant fact with respect to this disturbance had been made known. Hayden and Sholl (1923-2h) (l92h-25) pursued the theory with intent to add further to the data already accumulated by the afore mentioned prOponents. The results of these workers brought out the fact that in ll the very early stages of parturient paresis there existed a slight hypo- glycemia with blood sugar readings of 35 mg per cent compared with the normal mean blood sugar of 51.7 mg per cent. They went further to compile data on the blood sugar levels, making use of the Folinénu sugar test, on seven parturient paresis cases. The data based on their work are shown in Table A which clearly indicate the presence of hyperglycemia in all seven cases. 'w'idmark (1926) attempted to explain the hyperglycemia in parturient paresis as reported by ha den and Sholl (l923-2b) (l92h-25) on the basis of absorption of lactose from the mammary gland during gestation. hayden (192?), however, demonstrated a consistently low blood lactose, which did not support the theory postulated by hidmark. The work of Schlotthauer (1926), in which the data on the blood sugar levels in eight parturient paresis cases as shown in Table B, sup— ported the findings of hayden and Sholl (l923-2h) (l92h-25). Moussu and houssu (l92c) concluded that parturient paresis is not due to hypoglycemia. This conclusion was based on a mean blood sugar level of 61.0 milligrams per cent for a series of cows suffering from parturient paresis. This conclusion was substantiated by two fee 5; first, that there is present in this malady a glycosuria which is not consistent with a condition of hypOglycemia, and second, in this condition the secretion of milk is diminished thereby lowering the demand for glucose in the udder for the production of lactose. Little et al. (l92b) determined blood sugar levels on a normal cow at hourly intervals for a period of 17 hours. Their findings, TABLE A BLOOD PLAShA GLUCOSE OF COQS‘WITH PAnTUhlhhT PARLSIS Hayden and Shell (1923-2h) (l92h—ZS) Plasma Glucose Preinflation Post-inflation Date Case mg % Time of collecting blood (min.) No. 10-25 30—55 CO-bS 105-115 g; “2:3 mg 26 mg :36 mg: 3‘6 10/7/26 1 68.7 109.6 137.5 - - 10/10/26 a 70.7 .. .. _ _ 10/1h/26 3 e2.2 61.3 131.1 - - 10/23/26 u h5.0 73.3 67.1 - - 10/29/26 5 77.8 111.5 108.0 99.6 67.0 10/31/2e s 62.7 77.7 113.h 119.h 7b.? 1/13/27 7 u9.o 00.0 cu.o 62.1 Reinflated Average 62.3 - 85.5 100.6 93.7 80.6 13 TABLE B BLOOD PLASMA GLUCOSE OF COWS‘LITd MILK FLVLR Schotthauer (1920b) Cow Post partum Symptoms Blood Sugar Symptoms Blood Sugar No. hours after mg x 2h hours mg % calving before after after treatment treatment treatment 1 6 general 75.h7 normal 5h.60 paralysis 2 12 general 87.75 normal 72.95 paralysis 3 3 comatose 107.50 conscious bh.50 unable to rise h 36 comatose 0h.50 normal 02.83 5 2h comatose 109.20 normal 57.h0 6 7h comatose 109.20 normal - 7 3 comatose 88.10 normal 02.80 8 2h comatose 156.20 normal - 1h illustrated in Table C, showed a variation for individual samples of between Sb and 93 mg per cent. These findings were in accord with those of Hayden (1927), as shown in.Table D. Since the data thus obtained indicated a variation of blood sugar levels between fairly wide limits care should be taken when statements of normal or abnormal blood sugar levels are made. The data in Tables A and B indicate that in all but two cases the blood sugar findings in parturient paresis are well above the level at which symptoms of hypOglycemia would be expected to appear. Further support in opposition to the hypoglycemia theory was pro- vided by the work of Dryerre and Grieg (1928) in which they reported a high blood sugar in eight cases of parturient paresis. These workers stated, "based upon the results of our experiments and upon considera- tion of all available evidence, we formed the firm Opinion that milk fever is not a hypoglycemia.“ Subsequent to the work of Dryerre and Greig, Peterson gt El. (1931) attempted to reproduce the parturient paresis syndrome, with considera- tion given to the hypoglycemia theory, by injecting insulin. They succeeded in lowering the blood sugar to 17 mg per cent at which level the subjects were held for prolonged periods of time without producing in any case symptoms suggestive of paralysis or coma. This fact, they believed, should dispel any question as to low blood sugar being a cause of parturient paresis. Hypocalcemia Theory Throughout the early part of the nineteenth century, at which time investigators were occupied with the compilation of facts to substantiate TASLE C hOUhLX BLOOD sheen VLLbES FhOh A hOHhAL cow Little, 33 31. (192E) Sample No. T ime blood Sugar as per cent of glucose Food Taken -- General Notes 10 ll l2 13 1h 15 17 10 ll 3 h LL later at h .11 . A.h. A .i-z. hoon fflh. dag)? S .090 .090 clotted .05b 7 lbs. hay fed at 5:30 F.M. chewing cud Taken before feeding h lbs. cake given after 0 A.h. test 25 lbs. silage fed after 9 A.h. test 20 lbs. mangels fed at 11:30 A.M. u lbs. cake fed at 3:30 P.h. 10 TASLE D BLOOD SUGAR VLLUmS OF NORMAL COtS hayden (1927) Mg of Sugargper 100 m1 of Blood --. .‘~~-..n. n-~ Cow NO. Glucose Lactose Glucose and __ Lactose 1 52,2 00.0 52.2 2 u5.8 00.0 h5.0 3 50.0 00.0 56.0 b 51 .145 00 .O 51 J45 or disprove the hypoglycemia theory, certain workers engaged in blood analysis of parturient paresis cases, observed a consistently low blood calcium in all cases. Thus the attention of all workers was directed toward the hypocalcemia theory of parturient paresis. Little and hright (1925) (1926) were among the first to report on the blood calcium levels ‘1‘ of cows suffering from parturient paresis. inese men first established the blood calcium levels on normal cows (Table h). They found the levels to range between 9.9 to 10.h0 mg per cent. The work of the above men, directed primarily at the discovery of the cause for the significant fluctuation in the level of blood calcium in normal subjects, failed to account for this variation. Meigs gt 31. (1919) attempted to aCCOUnt for the variation in calcium levels by stating "The chief controlling factor is probably the concentration 0, of bicarbonate in the plasma. It is probable that the concentration of calcium tends to vary inversely with that of bicarbonate." Hart et 31. (192A) supported this hypothesis. The results of Huffman and Robinson (1926) were not in accord with the above findings. They noted that the day to day fluctuation in blood calcium was not accompanied by any appreci- able change in carbon dioxide. Conversely, they observed marked changes in carbon dioxide without any corresponding Change in blood calcium. These investigators also reported that there was no indication that calcium and carbon dioxide fluctuate inversel . In a later paper Huffman and Robinson (1926) made known the fact that the carbon dioxide capacity of the blood was lowered in parturient paresis, a fact that was later confirmed by Sampson.and hayden (1935). '.J ! I . ’CCR fl Tawfi v ‘oc —~.- .‘ ..... § --u- .- .‘F 7‘ .11.: U J-J~¢L-'_ LHIALD C: .!;':‘_'t.‘.J—. Uch : 0 fl‘ .¢_ ,1. N \‘: L-Uv :, Cu 6.4. \..,.., .— __ n '. - \‘ v- .\ . - A -._. n- ~~ _w a bu n3. .". ‘ var v :1"; k , n 1 n: -,. by (c. n lit... \ ) é ‘4 KL) 10 .:.O 10 .CO 10 .‘20 9 .5. (I; 10.20 10 .00 10 J40 19 With a normal mean blood calcium level established, it was possible to correlate the consistently low calcium levels found by the investiga- tors when blood analysis were confined to parturient paresis cases. . Blood calcium levels in parturient paresis cases as reported by Little et 31. (1925) (1926) are shown in Table F. There was a consistently low blood calcium in all cases with extremely low values appearing in the most severe cases. Convinced, as most workers were that calcium in the blood plays an important role in parturient paresis attempts were made to duplicate the syndrome experimentally. Symptoms of hypocalcemia were produced by Peterson et a1. (1921) by the intravenous injection of a 20 per cent solution of sodium citrate. Becker (1933) made some observations which appeared to shed some light on one possible prOphylactic approach. According to Becker forages grown on acid soils are deficient in calcium, some times, to the extent that animals feeding upon them call upon the skeletal re- serve of calcium to meet the needs of other tissues of the body. The addition of lime to soils for forage crOps is widely practiced and it is difficult to ascertain whether or not the incidence of parturient paresis would be still greater today with higher producing cows had the soil liming practice not been followed. Greig and Dryerre (1932) reported on the subcutaneous injedtion of 30 to 60 grams of calcium gluconate in five per cent solution imme- diately after parturition as a prOphylactic measure. Some authorities believe that prepartum milking in heavy producing cows tends to lower the incidence of the disease. Smith and Blosser (19h?) made some TABLE F BLOOD PLAShA CALCIUM IN CCmS SUFFERING FuOh PAhTUhlhKT PARESIS Little, 3} 3;. (192:) Cow Down Cow hormal Remarks Case No. (duplicate) (mean) (duplicate) (mean) mgjé he” 1 b.50 n.50 9,5 9,00 Typical Case - 9.70 2 b.15 h.35 9.h0 9.35 Typical Case h.50 9.30 3 6 .00 5 .85 7 .95 7 .95 Hild Symptoms 5.70 7.90 h 5.:0 5.35 10.50 10.h0 Typical Case 5.10 10.70 5 6.uo 6.25 7.60 7.co Mild Symptoms 6.10 7.60 6 6.90 7.10 - — ' Mild Symptoms 7.30 - 7 b.05 b.20 - - Typical Case h.35 - 8 h.10 b.10 10.30 10.h5 Very Severe Case )4 .05 10 .00 9 3.60 3.70 6.70 6.65 Typical Case 3.50 6.95 10 5.75 5.65 7.75 7.60 Typical Case 5.95 7.65 11 5.10 5.20 8.25 8.10 Typical Case 5.25 8.15 12 6.30 6.35 9.00 9.75 Typical Case 6.35 10.00 21 extensive observations which led them to the belief that prepartum milking does not tend to lower the incidence of this disturbance. Along the same line it has been the generally accepted belief that the immediate and complete milking following parturition tends to precipitate an attack. The work of Smith et 31. (19h8) failed to support this belief. Parathyroid Deficiency Theory Coexistent with the hypocalcemia theory is that of parathyroid deficiency which was advanced by Dryerre and Greig (1925) with primary intent to disclose whether or not a failure of the parathyroid could be responsible for the apparent lowered calcium metabolism. The part played by the parathyroid was not completely weighed. 'Wilson and Hart (1932), following a study made to determine the reason for the low blood calcium in parturient paresis concluded that it was caused by two possible factors; first,an unusually large demand on the blood for calcium which the calcium regulating mechanism, though properly functioning, is unable to cope with and, second, a failure of the calcium regulating mechanism to take care of the demand for calcium which normally occurs at freshening time. Dryerre and Greig (1925a) (1925b), during a study in which they studied the possible role of the organs of internal secretion in parturient paresis concluded that there was an increase of guanidine in the blood. Since the work of Paton and Findley (1917) revealed that the parathyroids regulate the metabolism of guanidine in the body it could, therefore, be concluded that the parathyroids play a part in the 22 disease syndrome. However, the work of Hayden (1929) revealed that the guanidine in the blood of parturient paresis cases differs very little from normal tended to discredit the supposed active part played by the parathyroids in so far as the guanidine hypothesis was concerned. The parathyroids have a definite controlling influence on calcium metabolism and may play a functional role in parturient paresis. The work of Remiovsky and Rubin (1950) indicated that the total calcium level of the blood is probably the result of the interplay of numerous factors which include; calcium intake, calcium absorption, calcium excreation, vitamin D economy, parathyroid gland function, and phosphate, bicarbonate, protein and pH levels. The relations which determine the distribution of the blood calcium between the ionic and the bound forms are largely unknown. Godden and Duckworth (1935) observed that, in addition to a blood serum calcium drop in all animals at the time of parturition, a fall in the calcium complexes accounted for the major part of the fall in total calcium in cases of parturient paresis. It appeared to them that the symptoms of true parturient paresis became acute only when the values for both adsorbable and non-adsorbable calcium were simultaneously at low levels. Duckworth and Godden (l9h0) were the first investigators to partition the serum calcium during the transient reduction in serum calcium associated with parturition. They found that ionic calcium generally increased and with it were usually found increases in protein bound calcium as parturition approached. Duckworth and Godden (1936) recognized that a close relationship existed between ionic calcium and protein bound calcium. They observed that where the level of ionic calcium is depressed there is an immediate 23 mobilization of protein bound calcium and conversely when the amount of free calcium ions increases a portion becomes combined with serum protein. Further study by these investigators led to the belief that inorganic phosphorus reduction was accompanied by a reduction in total calcium. Thus it became apparent that inorganic phoSphorus could play a part in the parturient paresis syndrome. Palmer et 31, (1930) observed that there is a decrease in inorganic blood phosphorus which is manifested on the day before parturition. The decrease in blood inorganic phosphorus amounted to as much as 3.2 mgs per cent. 'Wilson and Hart (1932) substantiated this finding. Hayden (1938) brought out the fact that both calcium and phOSphorus decline at parturition normally but drop still lower in parturient paresis. Hypocalcemia coma, as observed in parturient paresis, is explained by Green (1939) as a nervous endocrine disturbance in which hyper function of the anterior pituitary plays the most important part rather than hypofunction of the parathyroids. The generally accepted physio- logical mechanism for the control of plasma calcium is the parathyroid' gland operating in conjunction with the nervous system and drawing on the reserves of calcium in the skeleton. The other endocrine glands, notably the anterior lobe of the pituitary, also play an important part. The inflation of the udder tends to stimulate the sympathetic and the para-sympathetic nervous systems in a way to effect the parathyroid endocrine gland to assert itself and bring about a temporary rise in blood calcium sufficient to cause the pituitary gland and other endo- crine glands to resume their normal function. The work of Frei and 21L Demmel (1932) led them to believe that parturient paresis arises more from disorders of the endocrine system and the vegetative nervous system associated with parturition than from the onset of milk secretion. The veterinary profession and research workers have mastered the field of clinical diagnosis despite the fact that there are voids to be filled before a clear and unguestioned understanding of this baffling malady can be had. They also have at their command two practical therapeutic procedures that can be applied for the relief of afflicted subjects. They are the air insuflation of the udder, and the intravenous or subcutaneous injection of calcium gluconate. 'With this knowledge of satisfactory therapy and with it's immediate and preper application very few cases of parturient paresis are lost. The primary concern of the animal husbandryman is a knowledge of some prOphylactic measure that can be relied upon to prevent the disturbance. Vitamin D Administration as Preventative Hibbs 33 3;. (191m) (191mb) did some work which indicated that massive doses of vitamin D immediately preceding parturition and shortly thereafter‘brings about a rise in the level of calcium in the blood. The preliminary work accomplished by the above workers indicated that daily doses of 2,000,000 U.S.P. units of vitamin D in the form of irradiated yeast fed daily for two weeks previous to parturition and continued one week post partum raised the blood calcium and phOSphorus twice as much as when 1,000,000 U.S.P. units daily were fed. This fact encourages the supposition that with the feeding of still higher levels of vitamin D a pending attack might be forestalled. The higher levels h) \f! of blood calcium and phosphorus thus attained are accomplished at the expense of the skeletal framework. With res both ‘rom the (l‘ ct to the incidence of parturient par;sis Ir‘ , standpoint of seasonal occurrence and breed susceptibility, much has been said and the opinions are variable. hetzgtr and Harrison (193C reported a higher incidence of parturient paresis in winter than in summer. Similar observations were made by henierson (193t). however, hiobs (l9ho), who studied parturient paresis over a period of four years, found the incidence of parturient paresis from May through September was the same as in the period October through April on a per- centage basis. Smith (19h?) confirmed the findings of Hibbs. Metzger and Korrison (193;), henderson (1936), and Hibbs (19ho), as well as others not reported in this review, agree that the percentage of parturient paresis is highest in the Jersey Breed. Magnesium Narcosis Theory Godden and Duckworth (1935'), and Hayden (193:) noted that there was a slight rise in blood magnesium in parturient paresis. Their observations were substantiated by Sjollema and Seekles (1932). These workers were of the Opinion, however, that the rise in blood magnesium was not sufficient to account for the coma in parturient paresis as being due to magnesium narcosis as suggested by Klobouk (1932). Summary of the Review of Literature Early in the 5th century research workers and veterinarians were plagued by a disturbance in dairy cattle occurring at or immediately 26 after parturition. A satisfactory therapeutic procedure had not been discovered at this time. Different drugs had been used without success. Various practices, such as blood letting, had been tried with unwarranted success. Veterinarians and research workers grasped at all evidence revealed on post-mortem for a possible clue to it's cause. Theory upon theory was propounded. Prominent among the theories were those of cerebral anemia, cerebral congestion, apoplexy, bacterial infection of uterine origin, bacterial infection of mammary origin, anaphylaxis, auto-intoxication, hypoglycemia, magnesium narcosis, and hypocaléemia. hith the expanded knowledge of physiology and endocrinology many of the theories were proven to be unfounded. Nevertheless the painstaking work of the early investigators who propounded these theories laid the foundation for further study that carried over into_the 19th century and up to the present time. Little experimental support is offered for any of the theories except the hypoglycemia, and the hypocalcemia theories. Since in recent years several workers have conducted research work which diSproved the hypoglycemia theory, investigators have concerned them— selves with the theory of hypocalcemia. Mineral metabolism assumed an important position in the basic studies of parturient paresis at this time. The consistently low blood calcium and inorganic phOSphorus levels at parturition and during an attack of parturient paresis along with the dramatic way in which affected subjects respond to calcium therapy lends strong SUpport to the hypocalcemia theory. PrOphylactic measures that are entirely satisfactory in forestall- ing an attack have not been discovered. The more recent work, in which it was found that massive doses of vitamin D administered immediately before and for a short time following parturition increased the calcium blood level, points the way to a possible prOphylactic procedure. To be accepted as practical it must at the same time be found economical. The only recourse to the handling of this disturbance is through therapeutic measures. The udder inflation method which was accepted as satisfactory has now been replaced by the calcium gluconate injection, either subcutaneously or intravenously. The latter is generally preferred. N (7) OBJECT OF THIS STUDY Since it is an established fact that in parturient paresis there exists a condition of hypocalcemia, and since this metabolic disturb- ance occurs in well nourished and heavy producing cows, and further since the therapeutic procedure of the intravenous administration of ionic calcium is accepted as being effective in restoring parturient paresis cows to health cows to health concern in this study is, there- fore, given to the relationship of bound and ionic calcium in the blood of affected animals. .—_..__ __ . v‘ 7 v-' ,“- R .. .— . . . _ , .- _ WUJ»~-~.|---;J r-.~U-h——J in “7‘14"” #3 rnnqv1'as “:0 “44:7 1 qp+~ A“..1~ pa ‘.:-~. ,a‘- 9'“ -C ¢ .LA. U-\A_.- U t.-\'.‘.-‘.v .“UC-.~~‘— wok—U LVLvA a: G_:~« wk 9:1 -va UVSU ~$P <‘ . $ 9 F '- ’v" p L 4-,“ ~ 0- ...- n —. t0 Baua-J.lEI. v-3: I13?" :1 I‘:J‘.:_: Cl uOual ClOOl CaaL'Vl . :I.“ L.” lain“. .- "1 r.- [7" r- q c 1‘-\t {‘5 4" “TN 3 ' T‘ $ "‘J“ " Calcllm inc CON- Elev Cu lo? this Cflpcs- h:-€ v-15“ au rshaou aLu '. - r‘.‘ ,—, ‘,.' -.,;-- .. .. :,..u. n - 4 - . a‘v .. he cows selected for this study of parturient Varesls were uLOoc that were pres itEi for treatment to the Farm Veterinarv Service at (I; n . u "a r‘.‘ “I _ "1 _. ».‘_ ‘.- - ., o 'mn~'_fi v ~ R _ . -_ - -- 3. ‘ . CZlean Duct: bOllei’e. and Sic CCLS (ladle ) TCEII‘t‘SdnLt-C. the I1J18ucln, “a n~- ’.. ‘.‘ .- ‘ , ,- r -_,4 O -l. “‘1 :,,.. A r". ’ucrnadj, nyI‘SEill‘e, c.1111 uEI‘Scy DI‘CCuS Ol Cacole. fullOlelu 3. Carcl' immediately preceiing therapy a :0 ml IT) 0) FJ. U) {U H LL parturient par ample of blood was Obtained from the “ugular vein. A. anti-coagulant (n q ‘ material was not used. Following the collection of the blooc sample all 0 O k U) I: (I‘ ’3 ’D (+- ’1 (D m ted by the intravenous inject'on of 500 ml of a 23 per cent solution of calcium glueonate. The partition of calcium was accomplished by the following procedure: After removing aliguot samples for total serum calcium, inorganic phos- phorus, and serum proteins the serum was placed in the ultrafiltration apparatus shown in Figure l. The apparatus consists of a three fourths inch length of Visking "Kojax" cellulose sausage casing fifteen sixteenth of an inch in diameter closed at the lower end by a number 0 rubber stepper secured tightly b—r means f a rubber band, and attached at the nPPéJL-JI‘US b‘OLi hLCOVEhI OF UL'L‘itA-FT Trail \n L“ b) (\D O Lecend 30/32 inch ho Jax.Cellulose Casing Isotonic Saline V tabiliz C” (t) Isotonic Saline A ir Mercury Line to'Eater Pump 54‘ A.) 3O TAdLE I DESCRIPTION OF COhS'WITH PAHTUHILNT PARESIS 31 Remarks Cow No. Breed Age dears) 1 Jersey 6 Typical Comatose 2 Holstein 7 Typical Comatose 3 Ayrshire 9 Typical Comatose h Jersey 6 Typical Comatose 5 Jersey 6 Typical Comatose 6 Holstein 5 Typical Comatose 7 Jersey S Typical Comatose 8 Holstein 6 Typical Comatose 9 Holstein 6 Typical Comatose 10 holstein 5 Typical Comatose 11 Guernsey 6 Typical Comatose 12 Holstein 8 Typical Comatose 13 Guernsey 8 Typical Comatose 1h Holstein 6 Typical Comatose 15 Jersey 5 Typical Comatose 16 Jersey h Typical Comatose 17 ‘ Jersey 6 Typical Comatose 18 Holstein 12 Typical Comatose 19 Jersey h Typical Comatose 32 upper end to a short length of thick-walled rubber tubing through which is inserted a three inch length of glass tubing. The latter is mounted in a number 8 rubber stOpper which is fitted tightly into a 250 m1 wide- mouthed Erlenmeyer flask. Filtration is hastened by applying a partial vacuum to the flask inducing a moderate pressure gradient between the two sides of the membrane. The ultrafiltration sac containing the serum is not left Open to the atmosphere, but communicates instead with a flask of physiological saline solution through which air entering the system must pass. The purpose of this arrangement is to prevent concen- tration by evaporation by making use of the fact that normal saline has approximately the same vapor pressure as the serum. A second glass tube in the stOpper leads to the vacuum pump (an aspirator) by way of another container of physiological saline solution, the vacuum release apparatus, described by Erhard and Decherd (l9h2), and a mercury manometer. Use of the vacuum release apparatus allows for easy regulation of the vacuum applied at a rather constant level. In most cases only about 12 to 15 ml of serum was available for ultrafiltration. 'Nith a vacuum of 200 1 mm Hg five to six hours were required to obtain enough ultrafiltrate for the calcium determinations. Biuret tests for protein on the ultrafiltrates were invariably negative. Furthermore, the ultrafiltrates were always water—clear and did not foam on shaking. Both the total calcium and the ultrafiltrable calcium were determined by the Clark and Callip (1925) method which is a modification of the Kramer and Tisdall (1921) method referred to by Tisdall (1923). In the case of the serum ultrafiltrates it was found desirable to use a four 33 ml aliquot sample rather than a two ml one because of the very low calcium concentration encountered in sera from cows with parturient paresis. Duplicate analysis were carried out whenever the Quantity of serum and it's ultrafiltrate permitted. It was thought advisable to test the permeability of the Visking cellulose tubing to calcium. Two experiments were performed with this purpose in mind. In the first experiment a solution of calcium chloride adjusted to a pH of approximately six was subjected to the ultrafiltra— tion procedure described above. The ultrafiltrate and the original solution were then analyzed for calcium. In the second experiment a citrated bovine plasma sample was handled in a like manner. RESULTS The results for the partition of blood calcium from normal cows is shown in Table II. The results for the partition of blood calcium from parturient paresis cases are shown in Table III. The results of the work done to test the permeability of the Vik-~ ing cellulose tubing to calcium is shown in Table IV. The average total blood calcium for the normal cows computed from the results shown in Table II was found to be 9.9 mg per cent. The average levels of ionic calcium was 3.85 mg per cent. The average level of inorganic phOSphorus was 5.505 mg per Cent. The average level of total serum protein was 7.88 mg per cent. The average level of serum albumin was 3.6h mg per cent. The average total blood serum calcium for the parturient paresis cows shown in Table III was found to be h.29 mg per cent. The average leVel of ionic calcium was 2.126 mg per cent. The average level of inorganic phosphorus was 2.75 mg per cent. The average total serum protein was 7.28 mg per cent. The average serum albumin was h.3h mg per cent. A comparison of the average determinations taken from both Tables II and 111 showed a pronounced drOp in total calcium, ionic calcium, and inorganic phOSphorus in parturient paresis cases. The average total serum protein and serum albumin remained statistically constant both in normal cows and in cows suffering rom parturient paresis. Table IV shows computations that reveal the efficiency of the viking cellulose tubing as an instrument in the partitioning of blood serum calcium. The results of the two trials indicated that both calcium chloride solution, and citrated bovine plasma passed the membrane 1005. This fact justified the assumption that the levels shown in Tables II and III are truly representative. TADLE II PAOTBIN, Ahfl thUm ALhUmlN OF fiOdfinL COLS bLOOD SLLUH GnLCIUm PAhTITICN, lhOhGthC PHOSPHOnUS, TOTAL SERUM Total Ultrafilterable Calcium Inorganic TSP? ilB.i* Cow ho. Calcium mg k p total PhOSphorus mg % mg % mg % Calcium ‘hg t l 9.6 3.7 .38.5 o.o9 7.63 3.66 2 10.9 3.6 33.0 b.ho 7.80 3.16 3 9.b 3.9 hl.5 b.3h 7.99 3.61 h 9.0 5.1 S2.h 5.62 o.oo h.3o 5 10.1 h.l h0.3 b.9l 7.59 b.6u o 10.5 h.3 bl.l 5.70 7.75 n.56 7 10.3 h.3 h2.1 S.oo 7.L9 h.oh 8 9.7 3.h 35.1 5.32 8.1h 3.63 9 10.5 3.7 35.0 5.0h 7.9h 2.55 10 9.3 3.2 3b.? 5.25 9.38 2.b2 11 9.0 2.75 30.6 5.oo 8.03 3.70 12 io.u h.0 3o.5 b.05 8.05 b.08 13 9.9 h.0 h0.h 5.36 7.77 2.h3 * TSP '= total serum protein %% Alb. serUm albumin TAbLE III BLOOD SLdUh CALCIUM PAfiTITION, INOAAGLIC PnOSPHOnUS, TOTAL SERUM PhOThIN, and Sbfihh ALbUhIN OF PnnTUhIENT PidnSIS COaS Total Ultrafilterable Calcium Inorganic TSP? Alb.%* COW NO. Calcium lugia p total Ifiwsphorus Imgio mg g mg % Calcium mg % - 1 3.u 1.2 35.3 0.67 6.51 b.38 2 3.5 2.1 5t.o 5.12 6.26 h.19 3 us 1.6: lo .0 2.9o 7.153 3.73 h b.6 1.5 33.3 2.02 7.36 3.93 5 3.0 1.7 to.h 0.52 8.01 h.&7 o h.h 1.95 no.3 1.76 7.1o 3.ho 7 3.h 1.o5 5h.h 1.h5 6.b6 3.96 o 3.h 2.0 5o.c 1.3o 7.o1 h.21 ‘ 9 5.0 2.5 50.0 2.73 7.00 h.hl lO 3.2 1.3 u0.o 6.55 7.91 h.51 11 u.1 1.95 L7.o 5.o5 - - 12 u.5 2.55 50.7 2.ho 7.Lh h.ll 13 3.5 2.0 57.1 1.23 7.00 5.56 in 6.3 3.15 50.0 1.15 7.6h 3.o2 15 3.3 1.3 39.b 1.60 5.98 b.0h 1o h.2 2.3 5h.o 1.o9 0.8o h.oh l7 h.h 2.1 u7.7 3.10 7.03 - 16 u.0 2.15 53.8 b.77 6.05 h.83 19 h.1 2.5 o1.0 2.h2 - — % TSP 1‘ 1 'X’X‘ lilo . total serum protein serum albumin 38 TABLE IV TEST FOE meflxfilLITY OF VIKING CEILULOSL‘J TUBING chED IN THE CALCIUI'E PILdTITION PnOCrLDUILE Calcium Solutions Total Ultrafiltrable Calcium Tested Calcium ' mg :6 mg :6 :6 of total calcium Calcium Chloride 8.9 9.1, 102 Citrated bovine plasma 9 .h 9 .h . lOO 39 DISC‘SSION Two significant facts are apparent with regard to parturient paresis. First, there was a consistently low blood calcium in cows stricken with this disturbance. Second, there is an immediate and, in most cases, a dramatic recovery from symptoms following the administration of calcium salts by the intravenous route, The first of these facts is shrouded with mystery primarily because of still another fact which can be answer— ed only in the negative. ‘hhy in the case of a strong, vigorous cow, most certainly possessed of a vast store of calcium in it's tissues, does this depletion of the blood calcium occur to the extent that hypo- calcemia and the attendant paretic state exist? The second of the above facts is clearly understood only in part, The complete answer there, too, is likewise in the negative, hhy does the administration of cal- cium salts into the blood stream, apart from the fact that it's presence overcomes 0r corrects the hypocalcemia by the restoration of a normal blood calcium level, activate the physiological processes adequately to maintain the level of calcium without a recurrence of the original pathological state? The answer to all of the above, not too completely understood phenomona, has been attempted by statements of endocrine gland disfunc- tion or failure. Lack of any specific criteria to explain endocrine disfunction beyond the fact that the parathyroids are known to play an important part in calcium metabolism leads one to ask the following to Question. how can tissue disturbance or failure of the endocrine system be responsible in this case when failure of any such conseguence by the system has not simultaneously had it's untoward effects upon gestation itself? The most recent attempt to maintain a high level of calcium in the blood of the administration of massive doses of vitamin D (2,000,000 U.S.P. units or more) immediately preceding parturition having been successful in increasing blood calcium levels leads further to the be- lief that the endocrine glands responsible for calcium metabolism must be functioning adequately enough. True it can be said that this is all accomplished at the expense of the skeletal framework. but can it not also be said that even this shift of calcium store could not have been effected had the endocrine system not been functioning in a relatively satisfactory capacity? Any serious disturbance of any of the organs responsible for calcium metabolism can not exist, it is felt, for tissue changes of an extent to modify the normal processes sufficiently to bring on the grave symp- toms observed in this disturbance could not be repaired within the brief period of time that recovery takes place following proper and adequate therapy. The work accomplished in connection with this study brought out the fact that both protein bound calcium and the ionic calcium drOpped in parturient paresis. At the same time, it was noted, the ratio between protein bound calcium and ionic calcium remained relatively constant both in health and in parturient paresis. 'hhat is the significance of this relationship? The fact has been demonstrated that there is a continuous compensatory action existing between bound calcium and ionic calcium. This physiological truth deserves consideration. In parturient paresis there is a definite lack of muscular co- ordination. This loss of normal function involves both the voluntary and involuntary musculature. There must be at least one of two states existing. First, there could be a disturbance of the nervous system. Second, there could be a negative calcium balance within the muscles themselves. Szent-Gyorgyi (19h?) demonstrated a strong affinity between calcium and muscle tissue, and brain tissue. Could not a transitory shift of calcium from the musculature or from the brain tissues be responsible for this syndrome? The above mentioned worker observed that this high adsorptive power toward potassium and cations, including calcium, in general seems to be intimately connected with the function of myosin which is an active component of muscle. Brain tissue, likewise, has a similar affinity for calcium. It is not without reason to surmise that by the establishment of the normal levels of blood calcium some physiological process hastens to replenish the supply of calcium to the muSculature as well as to the brain thereby restoring normal reflexes. The adsorptive power of structural proteins for calcium, and other metals bearing a positive charge is great. The enzyme systems could be functioning in a way to account, either wholly or in part, for such a change. The cortical hormones of the adrenal gland, which have a profound influence on the develOpment and function of muscle, will also have to be fitted into the picture, Szent-Gyorgyi (l9h7). It seems logical to assume that further approach to the study of parturient paresis should stem from the therapeutic knowledge of the disturbance. £3 summr In this study the blood serum samples from 13 normal cows, some in the dry stage and others in various stages of lactation, were partitioned for levels of bound and ionic calcium. The values for the ionic calcium were found to range from 2.75 to 5.1 mg per cent with a mean of 3.55 mg per cent. The bound calcium values ranged from h.7 to 6.8 mg per cent with a mean of 6.10h mg per cent. The blood serum samples from 19 parturient paresis cows were like- wise partitioned for bound and ionic calcium. The values for ionic calcium ranged from 1.2 to 3.15 mg per cent with a mean of 2.12o mg per cent. The bound calcium ranged from l.h5 to 3.15 mg per cent with a mean of 2.082 mg per cent. A noteworthy fact revealed in this study is that the ratio between ionic and bound calcium remained relatively constant both in health and in parturient paresis. ht EifiLFn‘liifi‘IijS Allcroft, h. h. and n. a. Green. Blood Calcium and Magnesium of the Cow in health and Di ase . Biochem. J. 2c: 2220-222b, 193D. Auger, h. L. M 2cherches sur la pathogenic de la fievre vitilaire. Rev generals de med. vet. ’5: 53-—3o)%192o. (”lranslat 3d by P. A. Fish, Cornell Vet. 17: th— 73, 17'27). Ayrault. Cited by Thomassen. A Contribution to tLe Study of Parturient Paresis of the Cow. J. Comp. Path. and Therao. 3: 1-9, 1690. Billings F. S. The nelation of Animal Diseas see to Public health and their Plevcntion. Am. J. Vet. Med. b: to-bo, loch. Clark, E. P. and J. o. Callip. A Study of the Tisiall Method for the Determination of Blood Ser um.hith a Suggested Modification. J, biol. Chem. 63: L01, 1325. Cox, J. h. Parturient Apoplexy in Cattle Commonly Known as milk Fever. Vet. 573 193-197, lboh. 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J. 29: hhS, 1935. Green, a. d. Recent Advances in Our lnoled'e of Diseases associated w'ith.hinera13a1ance in the blood of huminants . heport of the rational Vet. Med. Assoc. of Great Britain and Ireland, London, 1939. Creig, J. R. and h. Drye rre Prevention of hilk Fever. Rec. med. vet de liecolea diulfort, 107: hOh-hOO, 1932. Guillebeau, A. On the Patholo:~ical hnatomy of hilkF rev ver. Vefi. J. 23: 97-102, 191g. hart, E. B., H. Steenbock and C. A. Elvehjem. Dietary Factors Influen