Urinary Estrogen Excretion Rates During Pregnancy in the Bovine1

Urinary Estrogen Excretion Rates During Pregnancy in the Bovine1

Urinary Estrogen Excretion Rates During Pregnancy in the Bovine R. E. ERB,2 R. D. RANDEL, 2 T. N. MELLIN, ~'~ and V. L. ESTERGREEN, JR? Department of...

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Urinary Estrogen Excretion Rates During Pregnancy in the Bovine R. E. ERB,2 R. D. RANDEL, 2 T. N. MELLIN, ~'~ and V. L. ESTERGREEN, JR?

Department of Animal Sciences, Purdue University, Lafayette, Indiana, and Washington State University, Pullman Abstract

Urine was collected from 48 cows from 101 to 285 days of pregnancy, to define urinary excretion rates of estrone, 17 flestradiol, 17 a-estradiol, and an incompletely identified triol, hereafter referred to as estriol. Enzyme hydrolysis was used as the first extraetion step to minimize destruction of 17 a-estradiol. The estrogens were separated and purified by paper partition chromatography and measured fluorimetrically. Breed, age, and live weight of the eow were not significantly related to excretion rate when the latter was adjusted to a 5(~0-kg basis ( ~ g / h r - live weight × 500 kg). Average total estrogen excretion _+_one standard error per hour per 500 kg during pregnancy was as follows: 101 to 123 days--300 ± 96 /~g; 165 to 175 days-581 ÷ 154 /x~o", 200 to 212 davs--489. ÷136 ~g: 226 to 237 d a y s - - 6 9 1 ± 104 /~g; 250 t . 254 days--l,809 ± 580 ~g: and 271 to 285 days--3,402 ± 412 ug. The latter two means were significantly higher than means of each of' the other periods. Estriol excretion was highest during 250 to 254 days (P < 0.01, except for 226 to 237 days). Changes in 17 fl-estradiol were not significant, but estrone increased significantly (P < 0.01) by 271 to 285 days and 17 a-estradiol increased significantly (P < 0.01) by 250 to 254 days. Excretion rate of estrone tended to be higher or equal to 17 a-estradiol through 237 days, but 17 a-estradiol increased tenfold thereafter, compared with a fourfold increase for estrone. As recently reviewed, relatively large quantities of estrone and 17 a-estradiol are present

in bovine urine during pregnancy (12). Also, generally minor amounts of 17 fl-estradiol and a fourth metabolite tentatively identified as 1,3,5 (10)-estratriene-3, 16fi, 17a-triol are present following intravenous administration of estrone-16J~C (9). This latter compound will be subsequently referred to as estriol, since it appears rather definitely a member of the triol group of endogenous estrogen compounds (9, 14, 16). Variations in methods used, especially those aspect involving hydrolysis purification, and specificity of the quantitative step, have resulted in large differences between laboratories relative to the reported amount of estrogens excreted in bovine urine during pregnancy (12). Formerly, hot acid was used to hydrolyze bovine urine for variable periods of time prior to extraction of the estrogens. I t was subsequently shown that 17 a-estradiol was especially acid-labile and that yields of this metabolite were increased if an enzyme hydrolytic procedure was used prior to initial extraction with ether (7, 9, ]4, 18). I t seems clear that urinary estrogen excretion rate increases rapidly during the last one-half of bovine pregnancy (12) and that a further significant increase occurs during the 40-hour period preceding parturition (15). The magnitude of the changes during pregnancy is not clear, because of the different methods employed (12, 15). I t was our purpose to measure urinary estrogen after 100 days of pregnancy, to study chang'es in excretion rate of the metabolites using a method that minimized destruction of 17 a-estradiol. A brief report has been made using the data unadjusted for body weight (5). Experimental Procedure

Urine was collected during 1961-65 at S-hour intervals for 24-hour periods from 12 Guernsey, Received for publication July 7, 1967. 1Journal Paper no. 3115, Purdue University 28 Holstein, and 8 Jersey cows from 101 to Agricultural Experiment Station, 47907, Projects 285 days of pregnancy, using the harness and 1306 (Indiana.) and 1585 (Washington). A con- bag apparatus designed by Gorski et al. (8). tribution from Western Regional Research, Proj- Urine was collected from six cows one to two ect W-49. days preceding parturition, four cows prior " Purdue University. to sacrifice at 250 to 281 days, and 38 cows pre3Present address: Merck Institute for Theraceding ovarieetomy at 103 to 251 days. At l~cutic l:Iesearch, t/ahway, New Jersey. each 8-hour interval urine volume was re4Washington State University. 416


corded and a constant fraction or aliquot was filtered through spun glass-wool. The aliquot was boiled for ten minutes and held at 5 C until the end of the collection period for each cow. Then a 24-hour aliquot was prepared, frozen, and stored at --10 C until assayed. In some cases, the samples were stored as 8-hour aliquots until assayed. Initially, assays were done at Washington State University and then transferred to Purdue University in 1962. Urine samples requiring shipment were transported by air express in a frozen state in the presence of solid CO~. The urine was assayed for estrone, 17 flestradiol, 17 a-estradiol, and an estriol, using the method of Mellin et al. (14). Proof of identification of the compounds and precision of the method have been reported in detail (7, 13, 14). In brief, before hydrolysis trace amounts of estrone-16-1'C and 17 fl-estradiol~'C were added to the urine for the purpose of estimating method losses. Due to chemical similarity, recovery of the latter compound was used to estimate method losses for 17 a-estradiol. No attempt was made to estimate tile losses of estriol due to method, because the exact chemieal configuration was not definitely known (9, 14, 16) and radioactive triols were not commercially available. Enzyme (fl-glueuronidase) was used for initial hydrolysis, followed by acid hydrolysis, to minimize loss of 17 a-estradiol and to maximize total yield of estrogen extracted (7, 9, 14, 18). Following partial purification, the estrogens were isolated, using paper partition chromatography, measured fluorimetrically, and corrected where appropriate for method losses and quantity of added radioactive standards. The corrected concentration of each estrogen was multiplied by the volume of urine voided during' the time period represented by the aliquot assayed, and divided by the number of hours involved to establish the average excretion rate per hour. Statistical techniques included step-wise regression using both the build-up and tear-down models (1, 17), analysis of variance (17), and a multiple range test for determining differences between means with unequal numbers of replications (10). Results and Discussion

Variables a)~eeting statistical interpretations. 'riLe estrogen measurements were initially analyzed to test for linear, quadratic, and cubic relationships during pregnancy, independent of breed, age, and live weight of the cow, using a tear-down, step-wise regression computer


program. This approach was unsuccessful, largely because excretion rate increased gradually from four to eight months and then accelerated (Fig. 1). Attempts to describe this change by various transformations were unsuccessful. Since averages for periods between 101 and 237 days and between 250 to 285 days appeared to be essentially linear with days of pregnancy, the two groups were analyzed separately. A build-up, step-wise regression program (1) was used, with the estrogen data both unadadjusted and adjusted for live weight of the cow (hourly excretion r a t e - live weight in kg X 500 kg). The latter procedure was the most satisfactory for the purposes of this analysis, since the partial regressions between urinary estrogen excretion rate per hour per 500 kg, and breed, age, and weight of the cow did not approach significance (P < 0.10). I n the data unadjusted to a 500-kg live weight basis, age of the cow was not a significant factor, but breed, stage of pregnancy, and live weight were confounded. Weight and stage of pregnancy were the two most important variables related to estrogen excretion rate in the data unadjusted to a 500&g basis. Excretio.~ rate (ttg/hr/500 kg). The averages and standard errors for excretion rates for the four estrogens and their total for six periods of pregnancy are shown in Figure 1. The two minor metabolites at each stage of pregnancy shown in Figure 1 were 17 fi-estradiol and estriol. The former did not vary signifietmtly (P < 0.10), but the period averages for estriol were significantly different (P < 0.01). Estriol excretion rate, 202 t~g/hr/500 kg at 251 days, was significantly (P < 0.01) higher than during the other periods, except 230 days (Fig. 1). Excretion of estrone was variable from one period to the next and did not show a significant (P < 0.01) rise until the 281-day period of pregnancy (Fig. 1). Though estrone excretion increased from 128 ± 15 t~g/hr at the 106-day period to 510 ± 143 t~g/hr at the 251-day period of pregnancy, one cannot be certain of the significance, because of the variation between cows. Excretion of 17 a-estradiol was relatively low through the 230-day period, but increased approximately fourfold by 251 days, as compared to the mean of the previous three periods and more than doubled again near the end of pregnancy (P < 0.01). Likewise, total estrogen excretion, though increasing from 106 to 230 days, was significantly (P < 0.01) higher during the 281-day period than during any of the preceding periods. At 251 days, total estrogen J. DAIRY SCIENCE VOL. 51, NO. 3

















~ ~

~T 1950


3462 ±











w 1800 _J



0(.9: 1 5 0 0 0


o t350 0 ~


~ 105C 3= UA

0_ 900


e: 75C (.9 o o


~_ 6oc 450



i01-123 = 106 n=13

165-175 x = 170 n=5

200-212 x = 205 n=7

226-237 x = 230 n=8

250-254 ~, = 251 n =5

271-285 X = 281 n = I0


FIG. 1. A v e r a g e


excretion rate of estrogen metabolites during pregnancy.

excretion was significantly higher as compared to 106-(P < 0.01) and 170-, 205-, and 230( P < 0.05) day periods of pregnancy. General di~c~ssio~. The most striking teature of these data is the sharp increase in excretion of estrone and 17 a-estradiol, especially the latter, during the last one month of pregnancy. Moreover, Mellin et al. (15) observed an additional 29% increase during the 40-hour period preceding parturition. The excretion rate during the 8-hour interval including parturition was 4,280--+ 695 # g / h r converted to a 500-kg live weight basis. This compares to 3,402-+-412 /~g/hr at 281 days and 1,809 ± J. DAIRY ~qCIEIWOEVOL. 51, NO. 3

580 /~g/hr at 251 days of pregnancy. Excretion of total estrogen during the 106-day period was approximately three times greater than a 90 /~g/hr rate reported for one 442-kg Holstein heifer one day before estrus (13). The failure to observe a greater change in excretion rate from the 170-day period through the 230day period is not understood. However, Veenhuizen et al. (19) reported that concentration of total estrogen in fetal cotyledons during pregnancy increased more between the periods 230 to 237 days (1.59 /~g/100 g) and 256 to 275 days than between the periods 102 to 158 days (1.03 /~g/100 g) and 230 to 237 days.


Furthermore, a threefold increase was observed f o r fetal cotyledons sampled after parturition (19). The total estrogen oresent in the fetal cotyledons increased tenfold from 102 to 275 days of pregnancy, due p r i m a r i l y to increased weight of the fetal cotyledons (19). Therefore, increases in u r i n a r y estrogen during pregnancy a p p e a r to coincide with increases in concentration of estrogens in the fetal cotvledons. The relatively low u r i n a r y excretion rate f r o m 200 to 237 days may p a r t i a l l v explain why cows do not ordinarily abort soon after ovarieetomy d i n i n g this period (6). Since there is evidence that extraovarian sources of progesterone are near a maximum during the 200- to 237-day period (3), the amount available m a y be sufficient to prevent sudden dominance f r o m estrogen, thereby avoiding abortion. This aspect is covered in more detail in a companion p a p e r (4). These interpretations of u r i n a r y estrogen excretion rate assume nearconstant ratios between feces and urine. I t has been shown that more than one-half of the estrogen is excreted in the feces in the n o n p r e g n a n t (9, 12) and p r e g n a n t (2, 11, 12) cow. The ratio of ~'C in feces and urine was nearly the same after administration of two dose levels of estrone-16-~4C during the luteal phase of the n o n p r e g n a n t heifer (9), and levels increase in both feces and urine during pregnancy (2). Acknowledg ments The authors gratefully acknowledge the assistance of R. B. Harrington and Mrs. Shirley Wolfe with the statistical analyses, and Mrs. Nancy Godfrey for the extraction and measurement of urinary estrogens in this and earlier studies (13, 15). References (1) Biomedical Computer Programs. 1964. W. J. Dixon, ed. Health Sciences Computing Facility. Dept. Preventative Medicine and Public Health, School of Medicine, Univ. of California, Los Angeles. (2) E1 Attar, T., and Turner, C. W. 1957. Speetrophoto-fiuorimetric Determination of Estrogen in the Urine and Feces of Cows During Different Stages of Pregnancy. Missouri Agr. Exp. Sta., Bull. 641. (3) Erb, R. E., Estergreen, V. L., Jr., Gomes, W. R., Plotka, E. D., and Frost, O. L. 1968. Progestin Levels in Corpora Lutea and Progesterone in Ovarian Venous and Jugular Vein Blood Plasma of the Pregnant Bovine. J. Dairy Sci., 51: 401. (4) Erb, ~. E., Gomes, W. R., Randel, R. D., Estergreen, V. L., Jr., and Frost, O. L. 1968. Effect of Ovarieetomy on Concentration of Progesterone in Blood Plasma and Urinary Estrogen Excretion Rate in


the Pregnant Bovine. J. Dairy Sci., 51:420. (5) Erb, R. E., Rande], R. D., and Estergreen, V. L., Jr. 1967. Urinary Estrogen Excretion and Levels of Progesterone in Blood Plasma of the Cow During Pregna~lcy. (Abstract.) J. Dairy Sci., 50: 1001. (6) Estergreen, V. L., Jr., Frost, O. L.. Gomes, W. R., Erb, B. E., and Bullard, J. F. 1967. The Effect of Ovariectomy on Pregnancy Maintenance and Parturition in Dairy Cows. J-. Dairy Sci., 50: 1293. (7) Gomes, W. l~., Mellin, T. N., and Erb, R. E. 1965. Comparison of Methods for Hydrolysis of Bovine Urinary Estrogen Conjugates. J. Dairy Sci., 48: 993. (8) Gorski, J., B]osscr, T. H., Murdock. F. R., Hodgson, A. S., Soul, B. K., and Erb, R. E. 1957. A Urine and Feces Collecting Apparatus for Heifers and Cows. J. Anita. Sci., ]6: 100. (9) Hunt, M. L., Legau]t, R. R., and Herrick, H. 1961. Metabolism of Estrone-16-C ~ in the Luteal Phase Bovine. Vth Biennial Sympos. Animal Reprod., Knoxville, Tennessee. [Cited by Mellin and Erb (12, 13).] (10) Kramer, C. Y. 1956. Extension of Multiple Range Test to Group Means with Unequal Numbers of Replications. Biometrics, 12: 307. (11) Levln, L. 1945. The Fecal Excretion of Estrogens in Pregnant Cows. J. Biol. Chem., 157: 407. (12) Mellln, T. N., and Erb, R. E. 1965. Estrogens in the Bovine--A Review. J. Dairy Sci., 48: 687. (13) Mellin, T. N., and Erb, R. E. 1966. Estrogen Metabolism and Excretion During the Bovine Estrous Cycle. Steroids, 7:589. (14) Mellin, T. :N., Erb, R. E., and Estergreeu, V. L., Jr. 1965. Quantitative Estimation and Identification of Estrogens in Bovine Urine. J. Dairy Sci., 48: 895. (15) Mellin, T. N., Erb, R. E., and Estergreen, V. L., Jr. 1966. Urinary Excretion of Estrogen by the Bovine Before and After Parturition. 5. Anim. Sci., 25:955. (16) Rommel, W., and Rommel, P. 1960. Ver gleichende quantitativ chemische Oestrogenbestdmmungen und Bioptische untersuchungen am Endometrium beim Rind in der indivuellen Puerperal-Oestruszyklus, und Graviditatsperiode (Zivischenkalbezeit). Aeta Endoerlnol., Suppl. 51:569. (17) Ostle, B 1963. Statistics in Research. 2nd ed. The Iowa State University Press, Ames, Iowa. (18) Sehott, E. W., and Katzman, P. A. 1964. Separation and Estimation of 17 a-Estradiol. Endocrinology, 74: 870. (19) Veenhuizen, E. L., Erb, R. E., and Gorski, J. 1960. Quantitative Determina,tion of Free Estrone, Estradiol-17 fl, and Estradiol-17 a in Bovine Fetal Cotyledons. J. Dairy Sci., 43 : 270. J. DAIRY SCIENCE VOL. 51, NO. 3