VARIATIONS IN SERUM CALCIUM, INORGANIC PHOSPHORUS AND MAGNESIUM LEVELS DUE TO STAGE OF LACTATION, SEASON, AND AGE IN AYRSHIRE COWS INJECTED WITH VITAMIN D 3 PRIOR TO CALVING Vappu Kossila, Paavo Niemelä and Erkki Koskenkorva Department of Animal Husbandry, University of Helsinki Received October 22, 1969 Lactational disturbances (parturient paresis, grass tetany, acetonemia) can be reduced to a minimum by proper nutrition and care of dairy cows. For instance parturient paresis, which is characterized by low serum calcium and phosphorus levels and often also by somewhat elevated magnesium levels, occurs mainly only in mature, aged cows, which are inefficient in calcium absorption from their feed, in mobilizing their bone stores, and in retaining their endogenous calcium balance (Hansard et al. 1954). Heavily lactating cows usually maintain a negative calcium and magnesium balance (Lomba et al. 1968, Paouay et al. 1968). The phosphorus balance, in turn, tends to be positive if the cows are fed according to their requirements except when they loose weight, which is usually the case during heavy lactation (ref. Stott 1968). Parturient paresis is not solely caused by drainage ofcalcium into the milk as was once believed, since serum calcium appears to be depressed (Niedermeier et al. 1949) and paresis may occur (ref. Stott 1968, p. 157) even in mastectomized cows at calving. Even though udder inflation alleviates the symptoms of paresis (Greig 1930) (decreases loss of calcium, phosphorus and proteins from blood to milk), colostral excretion of calcium in paretic cows is not higher than in normal cows (Hibbs 1948, Nurmio 1968). Grain supplement (Kendall et al. 1966), a high phosphorus-low calcium diet fed from 4to 6 weeks prior to parturition (Boda & Cole 1954), and feeding (Hibbs & Pounden 1955, Hibbs & Conrad 1960) or injections (Paloheimo 1968, Payne 1968) of vitamin D shortly before calving have proved beneficial in preventing milk fever. A negative calcium balance was noted before calving in cows developing milk fever at parturition (Ward et al. 1952). However, a high calcium diet does not prevent this disturbance (Boda & Cole 1954), https://www.c-info.fi/en/info/?token=PXKHpv6OAdMaE46k.utJKSXtL1kfxHiYW5uzTqA.4DhbODz9Rmo7hAlioiM-8WTHcUW67gPuscDBVe8KJghwIPUsO7E4FV0D6-_lFwK4TxkPNFGdT9TYbwn1UYVt3HpxcrNgRnF4yj7fmPyX_VDaghUasS9TcQuhC1AE393g78almFF8TCYW9JFiM6pKJvCKBae19_2Mv1x0j9ir1GdS0Kgnq3ULGzwl5AKkP4Vh5gqzPTMOLIoUm5sqOMbV10u34SCV 9 and in fact such a diet may provoke paresis in healthy cows (Albright & Blosser 1957, Ender et al. 1962). Stott (1968) believes that the amount of calcium and phosphorus mobilizable from the bones at calving is an essential factor, a high calcium-low phosphorus diet leading to a state of »bone starvation». A decrease in the appetite (Moodie & Robertson 1961) as well as in the efficiency of the intestinal absorption of calcium (Nurmio 1968, p. 90) at calving may also play a part in the development of paresis. Endocrine factors (thyroid, parathyroid, pituitary, adrenal cortical and estrogenic hormones) are apparently also involved in the development of lactational disturbances the incidence of which may be increased by excess or deficiency in the secretion rate of one or several hormones. The parathyroid hormone, which is important in the regulation of the plasma calcium level, was at one time believed to be of importance in the development of milk fever. How- ever, this hormone was found to be of dubious value in preventing and curing milk fever (Little &Mattic 1933). Jönsson (1960) failed to find any histological indication of para- thyroid insufficiency in paretic cows. Parathyroidectomized cows failed to develop milk fever at parturition (Stott & Smith 1957). Estrogens (Stott 1968) and thyrocalcitonin (TCT) (Barlet 1967, Young & Capen 1967) may be involved, both have properties that are contrary to the effects of parathormone. Estrogen titers are very high at the time ofparturition but rapidly decrease after the event (Turner 1966). TCT injected at a rate of 100 pg/min produced signs of milk fever in cows (Care 1968). Decrease in the number of secretory granules of the C-cells and in the TCT content of the thyroid observed in paretic cows suggest that there might be an acute discharge ofTCT at calving (Anast 1968). Due to effective physiological regulation systems in the body, the serum calcium level is not readily influenced by the intake of calcium from the diet. The inorganic phosphorus level in serum, in turn, is more readily affected by the amount of phosphorus in the diet and a shortage of this element eventually leads to a depression of the serum level. The serum magnesium level depends also on the continuous intake of this element from the diet, since the amount of mobilizable magnesium that can be stored in the cow’s body, is very small. Acute hypomagnesaemia is frequently seen in cows put on grass heavily fertilized with ammonium salts, especially if in combination with sulphur or potash, and sudden changes in environmental climate and feeding (ref. Rook & Storry 1962), as well as insufficient energy (Breirem & Hvisdten 1966) and sodium (Butler 1963) intakes, appear to increase susceptibility to this disorder. The purpose of this study was to find out, by means of investigating the serum calcium, inorganic phosphorus and magnesium levels, whether these levels: a) fluctuate during the pre- and postpartum periods in well-fed, high-producing Ayrshire cows injected with vitamin D prior to calving, b) are affected by the season of the year and by the age of the cow, and c) are indicative of possible shortages in the ratios of the cows in the herd under observation. Material During the indoor feeding as well as the grazing periods in 1963, altogether 207 blood samples were obtained from 41 either dry and pregnant or lactating, nonpregnant Ayrshire cows of the dairy herd of the Viik Experimental Farm (Tables 1,2 & 4). All cows had been injected with vitamin D prior to parturition. The entire herd of about 65 cows Table 1. Serum calcium, inorganic phosphorus and magnesium levels in Ayrshire cowsbefore and after calving (1963). Days pre partum Days post partum Serum 38—31 30-16 15—0 o—ls 16—30 31-45 46—60 61—75 75 < mineral (7) (11) (17) (34) (34) (31) (22) (22) (29) Camg% 9.85 ±0.19 9.21 ± 0.25 9.67 ± 0.22 9.33 ± 0.12 9.48 ± 0.10 9.63 ± 0.12 9.70 ± 0.15 10.00 ± 0.15 10.09 ± 0.10 P » 4.44 ± 0.31 5.20 ± 0.43 6.02 ± 0.42 6.15 ± 0.23 5.54 ± 0.26 5.31 ± 0.24 5.48 ± 0.22 5.16 ± 0.24 5.34 ± 0.23 Mg » 1.60 ±0.14 1.92 ±0.17 1.82 ± 0.09 1.67 ± 0.07 2.01 ± 0.08 1.69 ± 0.07 1.72 ± 0.09 1.64 ± 0.08 1.72 ± 0.07 Number of casesin brackets. All cows received vitamin D injections as explained in text. Table 4. Effects of seasonand ageon serum calcium, inorganic phosphorus, magnesium, blood hemoglobin and hematocrit values in Ayrshire cows (1963). Group I (younger cows) Group II (older cows) Month N C Ca mg % P mg % Mg mg % Hb Hc~ *N C Ca mg % P mg % Mg mg % Hb He April 16 9 9.54 ± 0.19 5.07 ± 0.24 1.71 ± 0.07 9.81 31.94 22 14 9.33 ± 0.16 5.04 ± 0.37 1.77 ± 0.09 9.80 32.47 May 17 9 9.68 ± 0.13 5.66 ± 0.10 1.45 ± 0.09 9.83 33.25 25 16 9.35 ± 0.09 5.47 ± 0.22 1.45 ± 0.06 10.12 32.90 July 21 17 9.92 ±0.12 5.95 ± 0.23 1.89 ± 0.16 10.10 34.12 23 17 10.03 ± 0.09 4.69 ± 0.25 1.66 ± 0.09 10.32 34.04 August 25 13 10.13 ± 0.09 5.58 ± 0.21 1.93 ± 0.07 10.36 33.86 16 12 10.22 ± 0.14 4.66 ± 0.16 1.76 ± 0.08 10.16 33.53 N = number of samples, C = number of cows, Hb = hemoglobin g/100 ml, He = hematocrit value. 11 Table 2. Serum calcium, inorganic phosphorus and magnesium levels in three cows with lactational disturbances (1963). Cow mg % (—1) (+ 19) (+33) (+4O) (+62) AINI Ca 7.82 8.22 8.74 9.62 9.34 P 6.21 9.95 7.35 6.40 6.03 Mg 2.53 3.72 2.55 1.95 2.07 (±22) (+36) PONSI Ca 4.81 9.14 P 10.53 8.92 Mg 3.53 1.70 (-7) (+6) (+7) (+ 14) ETTI Ca 9.02 8.62 8.94 10.02 P 6.40 7.56 6.30 5.80 Mg 1.14 0.12 0.49 0.61 Numbers in brackets indicate days before ( —) or after (±) parturition. Table 3. Changes in serum mineral composition during late dry and early lactation periods in control and vitamin D injected cows (1969). Days pre partum < 6 6—o Days post partum 6—lo 10—15Group o—s 16 < Vitamin D (13) (4) (7) (4) (4) (8) Ca mg % 9.48 ±0.12 9.69 ± 0.75 9.50 ± 1.28 10.33 ±O.ll 10.70 ± 0.01 10.72 ± 0.12 P » 5.28 ± 0.37 5.35 ± 0.65 4.25 ± 1.03 5.62 ± 0.55 5.94 ± 0.24 5.26 ±l.ll Mg» 1.20 ±O.ll 1.18 ±0.17 1.17 ±0.02 1.15 ± 0.02 1.16 ± 0.02 1.12 ± 0.02 Control (7) (2) (6) (6) (5) (6) Ca mg % 9.68 ± 0.65 10.28 ± 0.91 9.75 ± 2.03 9.37 ± 2.31 9.60 ± 0.68 9.32 ± 1.05 P » 5.26 ± 0.87 5.65 ± 1.70 3.88 ± 0.74 4.32 ± 0.70 4.52 ± 0.76 4.33 ± 0.55 Mg» 1.33 ±0.02 1.16 ± 0.00 1.24 ± 0.01 1.08 ± 0.01 1.04 ± 0.01 1.12 ± 0.01 Numbers in brackets indicate the No. ofsamples analysed. produced an average of 5095 kg milk and 232 kg butterfat during the control year 1962— 63, and 5416 and 249 kg respectively during 1963—64. During April-May in 1969, 72 additional blood samples were taken from 12 cows shortly before and after parturition (Table 3). 6 of the cows were treated with vitamin D and 6 were left as controls. The feeding and management of the herd has been recently described in detail by Kossila (1967). Parturient paresis has been prevented successfully by means of injecting vitamin D 3 intramuscularly twice into the cows, 5 million I.U. each dose, the first dose administered approximately 7 days before and the second dose on the calculated date of delivery (Paloheimo 1968). To prevent hypomagnesaemic tetany, the cows have received magnesium containing mineral salt mixture during the last two to three weeks of the stall-feeding period and during the entire grazing period. The summer in 1963, during which part of the data was obtained for this study (see Table 4), was unusually dry and from the beginning ofJuly the cows received hay twice daily in addition to the pasture grass. Thus the differences 12 in feeding conditions between the stall and the grazing periods were somewhat smaller than usually. Methods Jugular blood was drawn into the heparinized test tubes. The hematocrit (He) values were estimated by the jnicromethod using an International capillary centrifuge (14000 RPM, 5 y 2 minutes). The absorbance of hemoglobin (Hb) in the whole blood was read directly at 540 mp by a Beckman B spectrophotometer after first treating the blood sample with dilute ammonia solution. From the blood samples collected in 1963, the serum calcium was estimated by titrating the diluted serum sample (1 ml serum, 3 ml distilled water) with 0.01-N EDTA solution using murexide as an indicator. The total amount of calcium plus magnesium was estimated from the serum sample mixed with ammonia-NH4CI-buffer solution (pH 1Fi- 11.6) by titration with EDTA solution using eriochrome black Tas an indicator. The magnesium content was obtained from the difference (Mg + Ca) Ca = Mg. From the blood samples obtained in 1969 the serum calcium and magnesium values were estimated with an atomic absorption spectrophotometer and several calcium analyses were carried out simultaneously using the EDTA method, which tended to give some- what lower values compared to the atomic absorption method. The serum inorganic phosphorus was estimated according to Taussky & Shorr (1953), this method involving precipitation of plasma proteins with trichloracetic acid, addition of ferrous sulphate ammonium molybdate reagent to the supernatant liquid, and measuring the intensity of the blue colour with a Beckman B spectrophoto- meter at 840 mp. All analyses were carried out in duplicates. Statistical calculations were made according to Croxton & Cowden (1955). Results Effect of stage of lactation. The data obtained from 41 cows and con- sisting of 207 blood samples was divided into nine parts according to the stage of lactation as shown in Table 1. The mean calcium, inorganic phosphorus and magnesium levels in the serum of the cows during the nine stages of lactation are given in the same Table. The serum calcium level was lowest during the days 30—16 pre partum (9.21 mg %) rising to 9.67 mg %on days 15—0 pre p. During the days o—ls0—15 post p., the calcium level was quite low again (9.33 mg %), however, from this stage the calcium level increased consistently with adavancing lactation (the peak yield is usually reached between 35—45 days post p. in the herd) the increase from days o—ls0—15 to days 61—75 being highly signifi- cant (P < 0.001***). The inorganic phosphorus level, was relatively low during the earliest phase of the dry period (4.44 mg %), whereas relatively high values were noted during the days 15—0 pre p. (6.02 mg %) and o—ls0—15 post p. (6.15 mg %); thereafter a fairly significant (P < 0.02*) decrease to 5.31 mg % by the days 31—45 post p. (peak lactation) was noted. From this stage on, inconsistent non-significant variation occurred in the phosphorus level with advancing lactation. The serum magnesium level was relatively low during the first phase of the dry 13 period (1.60 mg %) the level increasing, however, before parturition. Even though the serum magnesium level as a whole tended to be lower in lactating than in dry cows, the highest mean value (2.01 mg%) was noted during the days 16—30postp., this value being significantly (P < 0.002**) higher than those obtained during other phases after calving (Table 1). None of the 41 cows exhibited signs of parturient paresis at calving even though two cows had a calcium level as low as 7.80 mg % (see cow AINI in Table 2). Definite signs of some lactational disturbance were noted in three cows during the indoor feeding period, i.e. in cow ETTI 5 days post p. and in the cows AINI and PONSI 18—20 days post p. The serum mineral values obtained for these cows are given in Table 2 and the case histories below: AINI (4th calving). Day 1 prep.: Ca level quite low. Days 17—19 postp.: Rothera test, urine + + +, milk + , scrum Ca slightly below normal, inorg. P and Mg elevated. Day 25: received Borocalcium Myrilas 200 ml, Dextrasol 500 ml and Ketocystin 750 ml intrav. Day 27: Dexa-Korti 10 ml intram. Day 40: cow appeared healthy, the milk yield had increased markedly, the serum mineral levels had returned to normal (Table 2). PONSI (sth calving) Day 20 post p.: Rothera test, urine ++ +, milk -f. Day 22: serum Ca critically low, inorg. P and Mg elevated. Day 26: Borocalcium Myrilas 200 ml subcutaneously into neck. Day 28: Borocalcium Myrilas 150 ml -f* Dextrasol 500 ml + Ketocystin 750 ml intrav. Day 30: Dexa- Korti 10 ml intram. Day 32: Dextrasol 500 ml intrav. Day 35: Borocalcium Myrilas PMD 400 ml -f Dextrasol 500 ml intrav. Day 36: serum Ca and Mg quite normal, inorg. P elevated. Within three days the appetite grew worse. Day 39: the cow was too weak to get up she was slaughtered. ETTI (3rd calving) Day 7 pre p.: serum mineral levels were normal. Day 5 post p.: signs of grass tetany, Rothera tests negative, cow was treated with Borocalcium Myrilas 120 ml intram., Borocalcium Myrilas PMD 800 ml -f Borocalcium Myrilas 200 ml intrav. Day 6: Ca and inorg. P quite normal, Mg greatly depressed, blood Hb (15.15) and He (48.5) somewhat elevated, received Borocalcium Myrilas PMP 400 ml intrav. + Digitalis 6 ml intram. Day 7: cow’s condition had improved, serum Mg had risen slightly. Day 10: appetite had decreased. Day 15: further normalization in the blood mineral levels. Day 16: body temp. 41 ° C, received penicillin, further decrease in appetite. Day 20: cow in poor condition, Rothera test, urine +-f, Day 21: cow was slaughtered, pathological changes were noted in heart, kidneys, liver, and intestines. Serum mineral changes in control and vitamin D treated cows. During stall feeding period in April-May 1969, a further study on serum cal- cium, inorg. phosphorus, and magnesium was carried out, in which six cows received two vitamin D injections both in conjunction with 1.5 million units of vitamin A and 250 mg vitamin E and six cows were left as controls. Both groups consisted of cows similar in respect of age, feeding, and level of milk yield. The results of this study have been summarized in Table 3. As Table 3 indicates, the control group has a somewhat higher mean serum calcium level during the dry period and during the period right after calving, but a lower level during the periods beginning from the 6th day post p. than the vitamin D group, respectively. In the latter, the serum calcium level increased consistently after calving whereas in control cows it remained more or less constant. In both groups the serum inorg. phosphorus level was markedly lower right after calving than during the late dry period. However, after calving the prepartum phosphorus level was rapidly regained in the vitamin D group whereas in the control group the level remained rather low (Table 3). 14 The serum magnesium level was similar in both groups and was slightly higher during the dry period than after calving. As a whole, the serum magnesium level was lower in this study (Table 3) compared to the results obtained earlier from the same herd (Table 1). The daily milk yield was somewhat higher (by 1.5—2.0 kg) in the vitamin D group than in the control group. Several cows in both groups had various disorders after calving. The type of disorder and number of cows suffering from it have been listed below: Disorder Control Vitamin D Retained placenta 4 2 Parturient paresis 1 1* Mastitis 3 2 Ketosis I*** 4** Lymphadenosis 1 vitamin D injections were given too early, the second one 2 weeks before calving two cows had previous histories of ketosis apparently secondary type of ketosis In addition to the above specifications, two cows in the control group and one in the vitamin D group gave birth to twins. An interesting case history of one cow in the control group is given below: JUOMA (6th calving April 4, 1969) calved three weeks too early, twins dead at birth (20 + 20