Effect Of Estrogen and Progesterone on Feed Intake and Hydroxyproline Excretion Following Induced Hypocalcemia in Dairy Cows1

Effect Of Estrogenand Progesteroneon Feed Intake and Hydroxyproline Excretion Following Induced Hypocalcemia in Dairy Cows1 L. A. MUIR 2,~, J. W. HIBBS, H. R. CONRAD, and K. L. SMITH Department of Dairy Science, Ohio Agricultural Research and Development Center, Wooster 44691 Abstract

The effect of the {emale sex hormones, estrogen and progesterone, and feed (calcium) intake on the ability of cows to replace blood calcium after induced hypocalcemia was investigated ha two experiments. Intravenous infusion of the disodium salt of ethylenediaminetetraacetic acid (EDTA) was used to induce hypocalcemia. Percent hacrease in urinary hydroxyproline excretion was the index of bone resorption. Neither estrogen injected at parturient levels nor progesterone at mid-pregnancy levels interfered with bone resorption, as thus indicated. Estrogen reduced feed dry matter intake, and thus calcium intake, whereas progesterone partially counteracted the effect of estrogen on dry matter intake. In both experiments multiple linear regression of the drop in serum calcium (mg/100 ml) due to EDTA infiasion, on (X1) calcium intake (g Ca/100 kg body wt/day) and (Xz) percent increase ha hydroxyproline excretion accounted for a significant portion of the variation. This demonstrated that the amount of calcium mobilized during hypocaleemia was dependent both on the quantity of calcium available for absorption from the gut and bone resorption, l~atios of the standard partial regression coefficients for calcium intake divided by percent increase in hydroxyproline excretion (1.35 and .78 for Experiments 1 and 2) indicated that intestinal absorption of calcium and bone resorption, as indicated by hydroxyproline excretion, were of

about equal impol~ance in preventing the decline in serum calcium induced by EDTA infusion. These results indicate that the major involvement of high blood estrogen at parturition in parturient paresis is not through inhibition of bone resorption but rather through decreased absorption of intestinal calcimn caused by estrogen induced inappetence; Introduction

Estrogen stimulated bone accretion in several species (12, 13). Ranney (12) found that estrogen stimulated bone accretion in mice while parathormone stimulated bone resorption and inhibited bone accretion. When ho administered estrogen and parathormone together, an antagonism was demonstrated suggesting that estrogen opposed the function of parathormone in promoting bone resorption. Stott (15) has suggested that the exceptionally high systemic estrogen at parturition could interfere with the action of parathormone on bone and predispose such cows to parturient hypocalcemia. On the other hand, progesterone with its anti-estrogenic activity might counteract the estrogen effect. From preliminary field data Payne (10) has demonstrated effective prevention of parturient paresis in cows dosed with progesterone. The purposes of this study were a) to test the hypothesis that estrogen at parturient levels interferes with resorption of calcium from bone, b) to determine the relative importance of dietary and skeletal calcium as sources of available calcium in adult dairy cows under the stress of induced hypocalcemia, and c) to study the extent to which progesterone might counteract the action of estrogen.

Received for publication January 6, 1972. Journal Article 104-71, Ohio Agricultural Be- Experimental Procedures search and Development Center, Wooster 44691. Experiment 1. Three adult, nonpregnant, Present Address: Merck & CO., Inc., Rahway, nonlactating Holstein cows received four horNew Jersey. a Data in this paper were presented to the mone treatments ha a three by four factorial Graduate Faculty of The Ohia State University design. Treatment of estrogen, progesterone, in partial fulfillment of the requirements for the both, or neither (control) were given to each degree Doctor of Philosophy. cow in random order, One month was allowed 1613

1614

M U I R E T .AI..,

TABLE 1. Hormone injection schedule for Experiment 1. Day

Estrogen

Progesterone

(rag 600 kg body wt/day) 1

2 3 4 5 6 7~

17

170

85 153 220 220 220 220

170 170 170 170 170 170

" Cows were infused with EDTA.

between treatments, During treatment cows were housed in metabolism stalls and fed their usual ration of concentrate plus alfalfa hay free choice. The hormones, 17 fl-estradiol and 4pregnen-3, 20-dione (Mann Research Laboratories, Inc., New York, N. Y.), were dissolved in absolute ethanol and given subcutaneously twice daily for seven days in amounts in Table 1. Feed intake and total urine excretion were measured, and urine samples were taken twice daily for two days before and two days after infusion of the sodium salt of ethylenediaminetetraacetic acid (EDTA). Cows were infused with EDTA solution for 6 hr on the seventh day of hormone treatment. Both jugular veins were eannulated with polyethylene tubing, and an infusion pump was used to deliver EDTA solution into the left iu~ular vein. A dose of 147 mg of EDTA per kilogram of body weight was infused during a 6 hr. Blood samples were from the right jugular vein prior to infusion and at hourly intervals during and for 2 hr after infusion. Additional blood samples were taken at various intervals during the following two days. Blood samples were analyzed for oxalate precipitable caleinm and urine samples were analyzed for hydroxyproline. Urine samples were filtered through cheese cloth, acidified with concentrated HC1 (25 v / v ) and stored at --23 C until analyzed. Experiment 2. Three adult, nonlaetating Holstein cows received supplemental calcium treatments in a three by three factorial design. Treatments of 8, 16 or 32 g calcium per 100 kg of body weight per day were given to each cow for 10 days. Cows received each ~xeatment in randomized order. Cows were fed a maintenance ration of concentrate and grass hay and were given a minimum of one month between treatments. Supplementary calcium was given as bonemeal in gelatin capsules twice daily during treatment to obtain the designated calcium JOURNAL OF DAIRY SCIENCE VOL, 55, NO, 11

intake and maintain a Ca:P ratio of approximately 2.2:1 in the entire ration. Feed intake and total excreta were recorded and samples taken twice daily during the last five days of treatment. On the eighth day of calcium treatment cows were infused for 6 hr with a solution of EDTA. The dose of EDTA infused was 147 mg per kilogram of body weight. The procedures for infusing EDTA and sampling blood were the same as described for Experiment 1. Blood samples were analyzed for oxalate orecipitable calcium and inorganic phosphorus. Urine samples were prepared as described in Experiment 1 and were assayed for hydroxyproline. Hay, concentrate, bonemeal and fecal samples were analyzed for dry matter, calcium and phosphorus. Analytical procedures. Urine hydroxyproline was determined by the procedure of Kivirikko et al. (5). One milliliter of urine was added to 1 ml of concentrated HC1, and the mixture was autoclaved at 120 C for 8 hr to hydrolyze urinary peptides prior to analysis. Feed and fecal samples were prepared for calcium and phosphorus analysis by wet ashing. One gram of sample was mixed with 20 ml of a 3:1, nitric to perchlorie acid mixhtre. The mixtures were covered and boiled to dryness. The residue was dissolved in deionized water and diluted to 100 ml. Blood serum calcium was determined by the Clark and Collip (2) modification of the Kramer-Tisdall method. Other calcium determinations were performed by atomic absorption spectroscopy by the procedures of Willis (17, 18) with certain modi~cations. Urine samples for atomic absorption analysis were diluted from 1:10 to 1:100 with deionized water and lanthanum chloride solution so that the samoles contained 5% lanthanum. Feed and fecal samples (1 g/100 ml) were diluted from 1:50 to 1:250 with deionized water and lanthanum chloride solution so that the samples contained .5% lanthanum. Phosphorus was measured colorimetrically by the Briggs (1) modification of the BellDoisy procedure. Results

Experiment 1. Figure 1 shows a typical response of the oxalate precipitable (unchelated) serum calcium of a cow to the 6 hr EDTA infusion. The unchelated serum calcium declined during infusion curvilinearly. After infusion serum calcium increased at a decreasing rate reaching the preinfusion level after about 40 hr. The degree of drop in serum eal-

CALCIUM MOBILIZATION

1615

- - •2 0 9-

~

>"

15-

E

g

8

>-~. I 0 -

v "~ Z

36

S-

o~ 5

16 24

EDTA

4046 54 64 72

8B

HOURS

1- INFUSION 1 I ! I I I 2 4 6 8 10 12

I 24

I 32

HOURS

Fro. 2. Effect of six hours ethylenediaminetetraacetic acid irffusfon on urinary hydroxypro]ine excretion.

17m. 1. Effect of six hours ethylenediaminetetraacetic acid infusion on setx~m Ca. einm during infusion is a measure of the cow's ability to mobilize calcium to replace the calcium chelated by EDTA (9). When bone is resorbed the collagen matrix, which contains approximately 14~ hydroxyproline, is broken down and much of the hydroxyproline is excreted in the urine. Therefore, urinary hydroxyproline excretion is an index of bone resorption. Figure 9. shows the effect of a 6 hr E D T A infusion on the urinary hydroxyproline excretion rate of a cow. Prior to infusion the excretion rate was relatively constant. During infusion the excretion rate was markedly elevated and after about i day

returned to the preinfusion level. The rate of urinary hydroxyproline excretion after infusion was highly correlated with the rate of hydroxyproline excretion prior to irffusion (r ----- .98). Thus, percent increase in urinary hydroxyproline excretion during the 24 hr following the initiation of EDTA infusion was used to express hydroxyproline excretion response to infusion. A summary of the data from Experiment 1 is in Table 2. The effects of hormonal treatment on percent increase in hydroxyproline excretion following ElYrA infusion, Table 3, shows that cows given estrogen alone had a higher percent increase in hydroxyproline ex-

Tam~ 2. Summary data from Experiment 1. Cow

I~

2b

3~

Treatment

Estrogen Progsterone EstrogenProgesterone Control

Dry matter intake

Initial HYP excretion

Increased HYP

(kg/day)

(rag/24 hr)

(~)

Ca intake

Serum Ca drop

(g/100kghody (mg/100ml) wt/day ) 5.7 7.77 .. 3.61

4.15 ..

235 228

65".4

8.20

217 354

69.1 4•.8

15.8 16.9

4.88

13.28

Estrogen Progesterone EstrogenProgesterone Control

10.50 12.75

605 586

43.1 54.8

18.5 23.7

4.69 4.45

11.52 ..

788 690

37.3 42.3

23.0 ..

3.83 2.77

Estrogen Progesterone EstrogenProgesterone Control

1.05 8.60

222 302

130.2 48.3

2.8 23.9

5.18 5.19

5.96 7.69

276 427

68.8 60.2

17.1 21.7

4.72 4.78

5.06

Body weight was 604 kg; 88.8 g of EDTA infused over 6 hr. b Body weight was 616 kg; 90.5 g of EDTA infused over 6 hr. Body weight was 596 kg; 87.5 g of EDTA infused over 6 hr. JOURNAl. OF DAIRY SCIENCE VOL. 55, N o .

iI

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MUIR ET AL

TABLE 3. Effect of estrogen and progesterone on percent increase in urinary hydroxyproline excretion during the 24 hr after initiation of EDTA infusion in Experiment 1. Increased Treatment

Orthogonal comparisons

Hydroxyproline

Estrogen Progesterone Estrogen-progesterone Control

88.4+16.5 ~ 58.0-+ 12.8 52.9_+12.8 45.7_+ 12.8

Comparison E versus P, C, EP EP versus P, C C versus P Remainder"

MS

P<

2141 2 228 492

.10

Standard error of the mean. b Remainder has 5 degrees of freedom. TABLE 4. Effect of estrogen and progesterone on daily dry matter intake prior to EDTA infusion in Experiment 1. Orthogonal comparisons

Dry matter intake

Treatment

Comparison

(kg/day) 5.2_+ 1,0• 10.6_+ 1.3 8.6 _-!-1.0 12.2 -+1.3

Estrogen Progesterone Estrogen-progesterone Control

E versus P, C, EP EP versus P, C C versus P Remainderb

MS

P<

56.81 13.92 2.56 3.@2

.02 .10

Standard error of the mean. b Remainder has 4 degrees of freedom. eretion than the other treatment groups ( P < . 1 0 ) . The percent increases in hydroxyproline excretion of cows receiving the other three treatments were not significantly different. The effects of estrogen and progesterone on daily dry matter intake are shown i n Table 4. Cows treated with estrogen ate less than the cows in the other three treatment groups

( P < . 0 2 ) . Cows given both estrogen and progesterone ate more than cows receiving estrogen b u t less thart control and progesterone treated cows ( P < . I 0 ) . Thus, estrogen had a significant depressing effect on dry matter (and therefore calcium) intake that was at least partially counteracted b y progesterone. Multiple linear regression analysis determined the effect of four independent variables

TABLE 5. Multiple regression analysis of drop in serum calcium (rag/100 ml) due to EDTA infusion on calcium intake, hydroxyproline excretion, estrogen and progesterone treatment in Experiment 1. X Variables Ca" Coefficient -- .194 SD .512 t Value 3.788° SPRCe 1.447 Equation Constant SE of Estimate F

Hydroxyproline~ -- .030 .009 3.497 ~ .852

Grams Ca consumed/100 kg body wt/day. ,bPer cent increase.

(P < .05). (P < .Ol). "Standard partial regression coefficient. JOURNAL OF DAIRY SCl/~NC~ VOL. 55, N o . 11

Estrogen -- .018 .015 1.190 .340

X Variables Progesterone .018 .017 1.099 .279

Ca ~ -- .318 .036 4.5144 1.028

Hydroxyprolineb -- .027 ,008 3.341 e .761 9.106 .538 10.737~

CALCIUM MOBILIZATION

1.617

TABI~ 6. Summary data from Experiment 2. Cow

Ca Intake

Ca Retention

Serum Ca drop

Fecal Ca

Initial HYP Excretion

1a

(g/100 kg BW/day) 8 16 31

(g/day) -- 17.~ -- 9.2 26.5

(rag/100 ml) 6.56 5,94 3.55

(g/day) 62.4 98.4 139.8

(rag/24 hr) 252 265 250

(%) 64.1 68.5 43.8

30.9 14.1 16.6

3.08 5.00 3.52

72.2 69.8 149.6

192 179 180

181.7 73.5 76.7"

--3.6

7.00

60.6

212

61.4

2b

8 16 31

3°

8 16

-

-

Increased HYP

Ago was 5 years; body weight was 538 kg; 79.0 g of EDTA infused over 6 hr. b Age was 8 years; body weight was 528 kg; 77.5 g of EDTA infused over 6 hr. * Age was 6 years; body weight was 705 kg; 103.5 g of EDTA infused over 6 hr. on the drop in serum calcium due to EDTA infusion (Table 5). The independent variables (X1 to X4) were estimated calcium intake (g Ca/100 kg body wt/day), hydroxyproline (percent increase in excretion), estrogen, and rogesterone. The dependent variable (Y) was e drop in serum calcium (mg/100 ml) due to EDTA infusion. The equation was: Y = 10.34 -- .194 X1 - - .030 Xz -- .018 X3 +

.018 X4

(SE of estimate = .54) [1] Only calcium intake (X1) and hydroxyproline (X2) were significant. Thus, a modified multipie regression equation was calculated with only these two independent variables. The equation was: Y = 9.11 -- .138 Xl -- .027 X2 (SE of estimate -----.54) [2] Equation 2 significantly fits the data (P<.05) and both independent variables were significant (Ca intake, P<.Ol, and hydroxyproline, P < . 0 5 ) . The standard partial regression coefficients for calcium intake and hydroxyproline excretion were 1.028 and .761. Experiment 2. A summary of the data from Experiment 2 is in Table 6. The influence of

calcium intake on percent increase in hydroxyproline excretion following EDTA infusion, Table 7, shows that cows fed the low dietary calcium (8 g Ca/100 kg body wt/day) had a higher percent increase in hydroxyproline excretion than cows fed the two higher levels of calcium (P<.13). With hydroxyproline excretion as an index of bone resorption, cows fed the low dietary calcium resorbed more bone calcium than cows fed the two higher levels of calcium. Multiple linear regression analysis determined effects of five independent variables on the serum calcium drop due to EDTA infusion (Table 8). The independent variables (X1 to Xs) were calcium intake (g Ca/100 kg body wt/day), hydroxyproline (percent increase in excretion), age (year), metabolic body size (kg .75), and apparent absorption of calcium (percent). The dependent variable (Y) was drop in serum calcium (rag/100 ml) due to EDTA infusion. The equation was: Y = 7.66 -- .I01 Xl -- .30 X 2 .073 X3 + .022 X4 -- .008 X 5 (SE of estimate ---- .31) [3] Equation 3 significantly fits the data (P<.05),

TABLE 7. Influence of calcium intake on poreent increase in hydroxyproline (HYP) excretion following EDTA infusion in Experiment 2. Orthogonal comparisons

Ca Level

Increased HYP

Comparison

MS

P<

(g/100 kg body wt/day) 8 16 31

(%) 102.4+19.7 a 52,8_+26,1 44.4 _+19.7

8 versus 16, 31 16 versus 31 Remainderb

5341 85 1168

.13

" Standard error of the mean. b Remainder has 3 degrees of freedom. JOURNAL OF ~DAIRY SCIENCE VOL. 55, NO. 11

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MUIR E T A L

TABLE 8. Multiple regression analysis of drop in serum calcium (mg/100 rrd)due to EDTA infusion on calcium intake, hydroxyproline (HYP) excretion, age, metabolic size and percent apparent absorption of calcium in Experiment 2. X Variables

Coe~eient SD t Value SPRCg

Ca ~

HYPb

-- ,101 .016 6.435 ~

(~) -- .030 .007 4.141 ~

.740

1.009

Age°

X VatSables BW ~°

Absa

Ca~

HY~

(~) -.o73 .148 •491 .074

Equation constant SE of estimate F

.022 .o13 1.760 .176

-,oo8

.OlO .837 ,134

-

.117 .o17 6.980 ~ .858

-- .033 .004

8.994 ~ 1.106 9.844 .398 43.765 t

a Grams Ca consmned/100 kg body wt/day. b Percent increase in hydroxyproline excretion. e Years. a Percent apparent absorption.

e (p < .05). (P < .01).

f Standard partial regression eoeffieient. but calcium intake (X1) and hydroxyproline excretion (Xz) were the only significant independent variables. Thus, a modified multiple regression equation was calculated with only these two independent variables. This equation was:

Y = 9.84 -- .117 Xl -- .033 X2 (SE of estimate = .40) [4] Equation 4 resulted in an equally significant and slightly higher "F" value for "goodness to fit". In addition the t values for X1 and X2 compared with Equation 3 (Table 8) increased. Regression coefficients in Experiment 2 are similar to those for Experiment 1. Results from both experiments indicated that the amount of drop in serum calcium during infusion was reduced when calcium intake was increased or when greater increases in bone resorption occurred as indicated by increased hydroxy'proline excretion. The standard partial regression coettqcients for calcium intake and hydroxyproline excretion were .858 and 1.106. Discussion

The preparturient level of estrogen in Experiment 1 was estimated from the data of Erb et aI. (3), who measured total urinary estrogen excretion in the bovine around parturition. Their data indicate that cows excrete about 196 mg of estrogen per 600 kg of body weight per day during 271 to 285 days after conception. Progesterone in Experiment 1 was based on the work of Raeside and Turner (11), who established that c o w s require a minimum of t00 mg of progesterone per 450 kg of hody weight per day to maintain pregnancy if the JOURNAL OF DAIRY SCIENCE VOL, 55, NO. I1

corpus luteum is removed. Based on these calculations, slightly higher l e v e l s , 220 and 170 mg per 600 kg of body weight per day, estrogen and progesterone, respectively, were used in Experiment 1. The hypocalcemia produced by EDTA infusion stimulates parathyroid glands of the cow to release parathormone (14) which, in turn, would bring about collagen degradation and resorption of bone calcium. Since collagen degradation leads to increased urinary hydroxyproline excretion (16), it was reasoned that EDTA infusion should be accompanied by increased paraflayroid activity and, thus, increased urinary hydroxyproline excretion. The data from these experiments showed that when EDTA was infused there was an increase in hydroxyproline excretion indicating that increased parathyroid activity accompanied by increased bone resorption did occur during and after EDTA infusion. Urinary hydroxyproline excretion rates before and after infusion were correlated. This showed that much of the variation in initial hydroxyproline was attributable to differences between cows. Therefore, the data were corrected for differences in initial hydroxyproline excretion rates. Expressing the data as percent increase in hydroxyproline excretion during the 24 hr following initiation of EDTA infusion rectified the data since most of the variation in serum calcium drop during EDTA infusion was accounted for when percent increase in hydroxyproline and dietary calcium intake were used as independent variables in multiple linear regression analysis.

CALCIUM

MOBILIZATION

1619

If estrogen had inhibited resorption of cal- dietary calcium as a source of available blood cium from bone in Experiment 1, hydroxypro- replacement calcium during hypocalcemia was line excretion would have been reduced by es- investigated further. Cows that received the trogen treatment. Orthogonal comparisons of low dietary calcium had a higher percent inthe means of percent increase in hydroxypro- crease in hydroxyproline excretion than cows line excretion showed that cows treated with fed the two higher levels (P<.13). This indiestrogen alone had higher percent increases in cated that cows receiving the low dietary calhydroxyproline excretion than cows in the oth- cium were required to resorb more calcium er treatment groups (P<.10). This indicated from bone, as were the cows treated with esthat cows treated with estrogen resorbed more, trogen in Experiment 1, because of the unanot less, calcium from bone during hypocaI- vailability of calcium for absorption from the cemia. In view of these results high estrogen gut. These observations support the concluat parturition wotdd not be expected to inter- sions of Luick et al. (6) that cows fed low calfere with the resorption of calcium from the cium diets were in negative calcium balance bone of parturient cows. but had larger mobilizable calcium reserves The difference in percent increase in hydro- that occurred at the expense of stable bone. xyproline excretion between cows treated with When multiple linear regression analysis was estrogen and cows given other treatments was used to test the regression of serum calcium considered physiologically important in view drop on the independent variables, calcium inof the observed effect of estrogen on feed take, percent increase in hydroxyproline ex(and, thus, calcium) intake. Estrogen treat- cretion, age, metabolic body size, and percent ment reduced dry matter intake (P<.02) apparent absorption, only calcium intake and thereby reducing the amount of calcium avail- percent increase in hydroxyproline were signifiable for absorption from the gut. Progesterone cant. The cows in Experiment 2 were of simiadministered alone did not affect dry matter lar age (5 to 8 years) and age may not have intake, but when progesterone was given with been distributed broadly enough to detect difestrogen, the reduction in feed intake caused ferences, or the variation due to age may have by estrogen was partially counteracted. Thus, been removed with the hydroxyproline index cows receiving both hormones ate more than of bone resorption. Dietary calcium intake and cows treated with estrogen but less than the the amount of EDTA infused were based on control and progesterone treated cows. Addi- body weight and hydroxyproline excretion was tional studies by Muir et al. (8) on the effects adjusted for animal differences by using the of estrogen and progesterone on dry matter in- percent increase in excretion. These adjusttake by cattle support these findings. ments apparently removed any variation due Depression of feed intake by exogenous es- to body size. Percent apparent absorption of calcium was not significant which suggested trogen has been observed in goats (4, 7). Meites and Turner (7) injected 4 mg of di- that the amount of calcium in the intestine ethylstilbestrol per day into goats and observed during hypocalcemia is more important than a loss of appetite and a reduction in fecal and percent apparent calcium absorption prior to urine output. Forbes (4) infused estradiol hypocalcemia. ( 1 2 0 / ~ / 2 4 hr) intravenously into goats and Standard partial regression coefficients indireported a significant depression in dry matter cated the relative importance of the independconsumption. ent variables. When the standard partial reResults of the multiple linear regression gression coefficient for calcium intake was dianalysis of the data from Experiment I showed vided by the standard partial regression coefthat the amount of calcium mobilized to re- ficient for percent increase in hydroxyproline place calcium chelated by EDTA was a func- excretion, a value of 1.35 is obtained for Extion of both calcium intake and bone resorp- periment 1 and .78 for Experiment 2. These tion. The amount of calcium available to re- values indicated that in Experiment 1 calcium place chelated blood calcium was elevated by intake was 1.35 times as important as bone reincreasing calcium intake while the higher per- sorption, as indicated by hydroxyproline excent hydroxyproline excretion values indicated cretion, in preventing the decline in serum calgreater calcium mobiliation from bone. In ad- cium due to EDTA infusion while in Experidition the results demonstrated that estrogen ment 2 calcium intake was only .78 times as and progesterone did not have a direct effect important. The reduced relative importance o f on the ability of cows to replace blood calcium calcium intake in Experiment 2 suggested that chelated by EDTA. although increased calcium intake helped cows In Experiment 2 the relative importance of replace blood calcium during hypocaleemia, JOURNAL OF DAIRY SCIENCE VOL. 55, NO, 11

1620

MUIR ET A L

higher dietary calcium was less efficiently utilized.

Acknowledgments The authors express their appreciation to Barbara Carson and Ted Ayers for their technical assistance.

References (1) Briggs, A. P. 1922. A modification of the Bell-Doisy phosphorus determination. J. Biol. Chem., 53:13. (2) Clark, E. P., and J. B. Collip. 1925. A study of the Tisdall method for the determination of blood serum calcium with suggested modifications. J. Biol. Chem., 63:47. (3) Erb, R. E., D. R. Randel, T. N. Mellin, and V. L. Estergreen, Jr. 1968. Urinary estrogen excretion rates during pregnancy in the bovine. J. Dairy Sci., 51:416. (4) Forbes, J. M. 1969. Effect of pregnancy of voluntary roughage intake in ewes. Abstr. J. Animal Sci., 29:157. (5) Kivirikko, K. I., O. Laitlnon, and D. J. Proekop. 1967. Modifications of a specific assay for hydroxyproline in urine. Anal. Bioehem., 19:294. (6) Luick, J. R., J. M. Boda, and M. Kleiber. 1957. Some biokinetie aspects of calcium metabolism in dairy cows. Amer. J. Physiol., 189:483. (7) Meites, J., and C. W. Turner. 1948. Studies concerning the induction and maintenance of lactation. II. The normal maintenance and experimental inhibition and augmentation of lactation. Missouri Agr. Exp. Sta. Res. Bull., 416. (8) Muir, L. A., H. R. Conrad, K. L. Smith, and J. W. Hibbs. 1970. Unpublished data.

JOURNAL OF DAIRY SCIENCE VOL. 55, NO. l I

(9) Muir, L. A., J. W. Hibbs, and H. R. Conrad. 1968. Effect of vitamin D on the ability of cows to mobilize blood calcium. ]. Dairy SCI., 51:1046. (10) Payne, J. M. 1970. Some recent work on the pathogenesis and prevention of milk fever, parturient hypocalcemia. Academic Press, pp. 1-13. (11) Raeside, R. I., and C. W. Turner. 1950. A preliminary report on the role of progesterone in the maintenance of pregnancy in the cow. J. Animal Sci., 9:881. (12) Ranney, R. E. 1959. Antagonism between estrone and parathyroid extract in their effects upon bone accretion. Endocrinology, 65:594. (13) Riggs, B. L., J. Jowsey, P. J. Kelly, J. D. Jones, and F. T. Maher. 1969. Effect of sex hormones on bone in primary osteoporosis. J. Clin. Invest., 48:1065. (14) Sherwood, L. M., G. P. Mayer, C. F. Ramberg, Jr., D. S. Kronfeld, G. D. Aurbach, and J. T. Ports, Jr. 1968. Regulation of parathyroid hormone secretion: Proportional control by calcium, lack of effect of phosphate. Endocrinology, 83:1043. (15) Stott, G. H. 1968. Dietary influence on the incidence of parturient paresis. Federation Proc., 27:156. (16) Weiss, P. H., and L. Klein. 1969. The quantitative relationship of urinary peptide hydroxyproline excretion to collagen degradation. ]'. Clin. Invest., 48:1. (17) Willis, J. B. 1959. The determination of metals in blood serum by atomic absorption spectroscopy. I. Calcium. Spectrochim. Acta, 16:259. (18) Willis, J. B. 1961. Determination of calcium and magnesium in urine by atomic absorption spectroscopy. Anal. Chem., 33:556.