REPRODUCTIVE HORMONES IN BLOOD AND URINE
OF OVARIECTOMIZED SOWS 1'2
B. L. Brown3 , R. E. Erb and R. D. Randel 4
Purdue University, West Lafayette, Indiana 47907
Summary ovariectomy, respectively. At day 245, 90% of
the total pregnanes was 5a-pregnan-3/3-ol-
Four yearling sows, averaging 170 kg body 20-one. Average excretion of urinary estrone on
weight, were ovariectomized 14 days after Day 245 was about 14% (3 #g/day) of the Day
estrus (Day 0) to study subsequent rates of 0 rate.
depletion of ovarian steroids in blood and their
Introduction
metabolites in urine for a period of 35 days.
Blood and urine were collected during estrus, 7 Blood and urine of prepuberal heifers
days after estrus, before (Day 0) and after (Day (Randel et al., 1971) and freemartins (Johnson
0.1) ovariectomy, daily through Day 14, and et aL, 1970; Randel et al., 1971; Wetteman et
then on Days 21, 28 and 35. Urine was al., 1972) contain ovarian steroids. Progester-
collected from three other sows 245 days after one has been measured in the adrenal venous
ovariectomy. Plasma total progestin, cortisol plasma of sheep, cows and pigs (Balfour,
and corticosterone were quantified by competi- Corrdine and Short, 1957) and in adrenal glands
tive protein binding and luteinizing hormone from cows (Erb and Stormshak, 1961) and
(LH) was measured by radioimmunoassay. sows (Erb et al., 1962). Estrone has been
Urinary estrone and four urinary metabolites of identified in adrenal glands of the cow (Beall,
progesterone (pregnanes) were quantified by 1939) but Gorski, Erb and Brinkman (1957)
gas liquid chromatography. Plasma cortisol and were unable to clearly establish the presence of
corticosterone were significantly ( P < .025) estrogenic biological activity in urine of
increased only at Day 0.I after ovariectomy. ovariectomized cows. Endogenous levels of
Plasma LH levels increased (P < .005) by Day 1 estrogenic and progestational hormones appear
and remained elevated through Day 35. Plasma insufficient to regulate luteinizing hormone
progestin (ng/ml) decreased from 18.7 -+ 6.3 (LH) levels in venous plasma from prepuberal
ng/ml on Day 0 to 4.8 + 1.9 on Day 1 and to heifers and freernartins (Randel et al., 1971)
1.8 -+ 0.2 on Day 2. Thereafter, no further and castrates (Taleisinik et al., 1969; Short et
decreases were detectable. In contrast, urinary al., 1973). However, nonovarian sources of
pregnanes decreased only 50% by Day 1 and these hormones may alter interpretation of data
did not decrease to estrous levels until Days 5 from intact animals when assessment of the
to 6. Average daily excretion of total urinary ovarian contribution is a primary objective.
pregnanes was 7.6 rag/day 14 days after estrus The objectives of this study were to define
(Day 0) compared to 2.4 mg/day at estrus and the effects of ovariectomy during the ovarian
0.48 and 0.17 mg/day 35 and 245 days after luteal phase of sows on rates of depletion of
ovarian steroids in blood and urine and to
l Journal Paper No. 5401, Purdue Agricultural determine the related effects on levels of
Experiment Station. A contribution from the
Department of Animal Sciences. cortisol, corticosterone and LH in plasma. A
2Tile authors thank Dr. G. D. Niswender, brief report has been made on LH (Brown et
Department of Physiology, Colorado State University, al., 1970b).
Fort Collins, for measuring blood plasma LH.
SPr~sent address: Department of Population
Dynamics, The Johns Hopkins University, 615 N. Experimental Procedure
Wolfe Street, Baltimore, Maryland 21205.
4Present address: U.S.D.A. Range Livestock Four yearling Hampshire x Yorkshire cross-
Station, Miles City, Montana 53901. bred sows weighing 160 to 180 kg were
606
JOURNAL OF ANIMAL SCIENCE, vol. 39, no. 3, 1974
HORMONES IN BLOOD AND URINE OF SOWS 607
ovariectomized 14 days after estrus. The sows, table 1) were generally higher but within 1.5
prior to and during the experiment, were fed, standard errors of the averages reported by
once daily, 2.7 kg of a mixture of corn and Tillson, Erb and Niswender (1970). The
soybean meal, containing 13% crude protein. averages at comparable periods of the estrous
Vitamins and minerals were added at recom- cycle were generally higher in the present
mended levels (NIR.C., 1964). Samples of study. Variation between individuals regarding
blood and urine were collected during estrus, 7 plasma progestin and urinary pregnanes was
days after estrus, once before and once after relatively large on the day of ovariectomy.
ovariectomy (14 days post estrus) and then However, by Day 2, compared to preceding
daily for 14 days. Thereafter, sampling was levels, average plasma progestin decreased (P <
done at 7-day intervals until 35 days postova- .005) to a level indistinguishable from subse-
riectomy. No ovarian tissue was found in any quent levels through Day 35 after ovariectomy.
sow when slaughtered. In addition, urine was In contrast, excretion of urinary pregnanes,
collected from three other sows 243 to 247
although decreased about 50% by 24-hr. after
days (X = 245) after ovariectomy (courtesy of ovariectomy, did not decrease to the low
Dr. B. N. Day, University of Missouri) for excretion rates observed (table 1) or expected
subsequent assay of pregnanes and estrone. during estrus (Tillson et a t , 1970) until Days 5
Anterior vena cava blood, collected by to 6 after ovariectomy (figure 1). There was
venepuncture into plastic bottles containing more than a 10-fold decrease in plasma
heparin, was immediately chilled in ice water progestin between the day of ovariectomy and
and then centrifuged at 0 C. The supernatant 2 days later as compared to a three- to fourfold
plasma was stored at -23C. Urine was collected decrease in excretion of urinary pregnanes
and processed as described by Edgerton and (table I; figure I). More rapid decreases in
Erb (1971a).
plasma progestin as compared to urinary
Quantitative methods used for assays were as pregnanes confirms earlier observations in
follows: plasma total progestin - Randel et al. intact sows undergoing luteal regression prior to
(1971), except the chromatographic and estrus (Schomberg et al., 1966a; Tillson et al.,
recovery steps were omitted; plasma cortisol 1970).
and corticosterone - Randel et al. (1971);
plasma LH - Niswender et al. (1969); urinary Although no significant decrease in rate of
creatinine - Hawk, Oser and Summerson excretion of total pregnanes occurred between
(1954); and urinary estrogens and metabolites Days 9 to 10 through Day 35 after
of progesterone (pregnanes) - Edgerton and ovariectomy, average rates decreased from 138
Erb (1971a). Quantification was done by +-- 19 to 88 _+ 16 ng. Urine collected from three
competitive protein binding (Murphy, 1967) other sows 245 days after ovariectomy averaged
for all plasma steroids, by radioimmunoassay 31 + 9 ng total pregnanes of which 28 + 10 ng
for LH and by gas liquid chromatography for was 5a-pregnan-3/3-ol-20-one. Levels of the
urinary steroid metabolites. Estrone trimethyl- other urinary pregnanes, 5/3-pregnan-3a-ol-
silylether was used as the internal standard 20-one, 5~-pregnan-3o~, 20~-diol and 5/~-preg-
during gas liquid chromatography of urinary nan-3a, 6a-diol-20-one (not shown in figure
pregnanes instead of cholesterol trimethyl- 1) were too low in each of the three sows for
silylether as described by Edgerton and Erb accurate quantification. The latter compound
(1971a). Excretion rates of steroids in urine also was low and generally undetectable in
were expressed as the ratio, nanograms per urine collected prior to and for 1 to 35 days
milligram of creatinine (Erb et al., 1970) and in after ovariectomy. This result was predictable
the text hereafter will be abbreviated to since Tillson et al. (1970) reported low and
nanograms (ng). Statistical procedures included frequently nondetectable amounts of this
analyses of variance and covariance as described pregnanediol in urine of nonpregnant sows
during the estrous cycle.
by Snedecor (1956). The Kramer (1956) test
was used to test for significant differences Edgerton and Erb (1971b) have suggested
between averages. that a portion of the urinary compounds
identified as metabolites of 14C-progesterone
Results and Discussion (Schomberg et al., 1966b) may originate from
the adrenal. Based on the data from sows
Progestin. Average excretion of pregnanes in ovariectomized 245 days, only 5a-pregnan.
urine and average plasma progestin during 3~-ol-20
estrus and on Days 7 and 14 after estrus (14, 7 steroids in consistently detectable amounts.
and 0 days preovariectomy, respectively in The parent adrenal steroid could be progester-
608 BROWN, ERB AND RANDEL
L~
+ l + l ~ +l+l+l+l
+1+1+1+1+1 ~+1~+1
r,~
~ + 1 ~ +1~+1~
~ +l +1 +i +J ~ ~ +1 +1 +1
+l+l+l+l ~ + 1 ~ +1
0
O~
~+l+l+l~ +l+l~+l
b-"
4 ~H +I 4-I 4"I +I "H +I -H 4-I
im
~;q; . o
[..., p 9~a0
ooy~
iai.
i
' ~.e~
HORMONES IN BLOOD AND URINE OF SOWS 609
TOTAL PREGNANES The relatively slow excretion of metabolites
60O ,t.--STANDARO ERROR of progesterone as compared to rapid decreases
in plasma progestin after ovariectomy during
5O0
"i the luteal phase may occur because of uptake
400 ~
lln
and release of progesterone or its metabolites
300
from the depot fat (Zander, 1961 ; McCracken,
2OO
1964). The storage of these compounds in fatty
I00
tissues may be of physiological significance,
h,
0 .i fl fl fl , particularly if animals differ markedly in
5.8-PRF'GNAN - 3Qo 2 0 ( / - DIOL
Z200.
Z fat-to-lean ratios. Based on covariance analysis
1.o there were no significant differences between
h i I00
sows in the rate of decrease in excretion of the
fin
n"
0 5O pregnanes from 3 to 35 days after ovariectomy
N r t] rh rh _
but the adjusted means were significantly
i
5 ~ - PREGNAN - 5 a - OL - 2 0 - ONE"
150
different for 5t~-pregnan-3/3-ol-20-one (P < .01),
5/3-pregnan-3a, 20~-diol (P < .005) and total
pregnanes (P < .005). The toregoing analysis
:~ 5o I
In suggests differences between sows regarding the
total quantity of progesterone to be cleared
'.0t 5 a - PREGNAN - 3 ~ - OK - 2 0 - O N E
from the tissues 3 days or more after
ovariectomy but no real difference in relative
150 rates of clearance. The sows were not markedly
different in degree of fatness by visual
50
0
2 5-4
nnnn
5-6
DAYS
7-8
AFTER
9-10 11-12
OVARIECTOMY
13-1g; 21-55
, 245
appraisal, a subjective measure which would be
too inaccurate to infer that differences in
fat-to-lean ratio did not contribute to the
significant differences between adjusted means.
Figure 1. Relationship between time after ova- A greater volume of data would be required to
riectomy and decreases in rates of excretion of make inferences about variations between
metabolites of progesterone (pregnanes) in urine of individuals in rates of secretion of steroids from
SOWS.
nonovarian tissues.
Urinary Estrone. No significant changes were
one since sow adrenals do contain the hormone detected in average excretion of estrone before
(Erb et al., 1962). Average daily excretion rates ovariectomy and for 35 days afterwards. The
of total urinary pregnanes prior to and after excretion rate already was low at the time o f
ovariectomy were calculated using an average ovariectomy which may, along with limitations
creatinine excretion of 1.35 mg/kg of body- of sensitivity of the method, explain the failure
to observe a significant decrease (table 1). The
weight (Erb et al., 1970) and an average weight
of 170 kg for the sows. One hour preceding three sows ovariectornized for 245 days
surgery, on Day 14 of the estrous cycle, the averaged 0.5 + 0.2 ng as compared to 1.2 + 0.3
estimated rate of urinary excretion of total for 4 sows 8 to 35 days after ovariectomy. A
pregnanes was 7.6 mg/day as compared to 2.4 0.5 ng/mg creatinine excretion rate would
mg/day during estrus and 0.48 and 0.17 mg/day translate to a daily secretion of about 3 /ag
35 and 245 days after ovariectomy, respective- using 86% as the expected urinary recovery of
ly. The latter rate was only 2.2% of the Day 14 metabolized endogenous estrogen (Brown et al.,
rate and 7% of the rate durin~ estrus. Since 1970a). Ttfis amount, if representative of
48.5% of intra.venouslyinjected C-progester- nonovarian tissue secretion, would be about
one was recovered in urine by Schomberg et at 14% of the rate observed on Day 14 after
(1965), the total pregnane excretion of 0.17 estrus. It is possible that this quantity of
mg/day 245 days after ovariectomy probably estrogen could have some biological effect,
represents only one-half of the parent hormone particularly if further study reveals differences
secreted from nonovarian tissue. If the parent between individuals not found in the present
compound was progesterone it appears unlikely study.
that such a low rate of secretion when added to Plasma Corticoids. Average plasma cortisol
the ovarian secretion would significantly affect was significantly ( P < .005) increased at 0.1
reproductive control mechanisms in the intact day after ovariectomy as compared to all other
nonpregnant sow. times except during estrus (table 1). Average
610 BROWN, ERB AND RANDEL
3G
28
to 4) probably reflected LH storage due to
~2.6
~- 2.4
~ STANDARD ERRO(~ progesterone inhibition of synthesis and release
during the luteal phase preceding ovariectomy.
~22 Post ovariectomy, day-to-day changes in sow
2.(~ plasma LH were much less random as compared
:2:
to cows (Brown et al., 1970b). Patterns of
change for individual cows from 1 to 42 days
.J
ED 1.4 after ovariectomy suggested a pnlsatlle type of
Z 12
LH release as in the ovariectomized ewe (Butler
et al., 1972). Geshwind (1972) has noted that
,~ 0 8
levels of LH in sow plasma changed less
abruptly, were not generally as high during the
0.2
estrous cycle and lower after ovariectomy as
0 compared to the cow.
0 0.1 6 7 8 I0 12 14 21 :~8 35
DAYS AFTER OVARIECTOMY
Figure 2. Relationship between time after ova- General Discussion
riectomy and levels of plasma luteinizing hormone in
sows (Day 0 was 14 days after estrus). Metabolites of endogenous estrogen and
especially progesterone attributable to ovarian
secretion were clearly detectable in urine of
plasma corticosterone at 0.1 day after ova- sows for 35 days after ovariectomy and a
riectomy was increased (P < .025) as compared further decrease in excretion rate was observed
to Day 0 and Days 1 to 35 after ovariectomy. when Day 35 rates were compared to Day 245
High average plasma cortisol during estrus (32 -+ rates. A depletion time exceeding 35 days was
1.5 ng/ml) may have been due to stress from unanticipated when the experiment was
handling the first day on experiment rather planned. Moreover, based on earlier data
than association with estrus. It has been shown regarding the adrenals as a source of estrogen
that levels of plasma corticoids decrease in and progesterone (Balfour et al., 1957; BeaU,
sheep as they become accustomed to handling 1939; Erb and Stormshak, 1961; Erb et al.,
(Bassett and Hinks, 1969) or following repeated 1962), higher excretion rates of their metabo-
short periods of fasting (Purchas, 1973), and lites in urine were expected than were observed,
that stress in heifers causes greater variability in especially in sows 245 days after ovariectomy.
plasma cortisol than in corticosterone (Willett At the latter time, sensitivity of the gas liquid
and Erb, 1972). No obvious effects of chromatography method was too low for
ovariectomy on plasma corticoids other than consistent detection of the compounds. Neither
the immediate effect of surgery was observed specificity nor sensitivity of the competitive
for any individual. If changes occurred because protein binding method was adequate to detect
of reduced secretion of ovarian steroids, these decreases which probably occurred in plasma
would have been masked by other undefined progestin 3 or more days after ovariectomy.
stresses which may have occurred during the The present data, involving only seven
experiment. ovariectomized sows do not prove that
Plasma LH. Average plasma LH increased nonovarian sources of estrogen and progester-
significantly ( P < .005) within 24-hr. after one would never be a factor influencing
ovariectomy (figure 2). Rayford, Brinkley and reproductive competence of individuals of this
Young (1971) reported that serum LH levels in species. Other factors such as environmental
sows were 2.4 -+ 0.4 and 2.6 + 0.3 ng/ml 24 and stresses, age and breed would have to be
52 days after ovariectomy as compared to 1.8 studied. The data do illustrate that metabolites
ng/ml during the estrous cycle ( P < .05). of progesterone and presumably tissue proges-
Although these averages exceed those shown in terone as well, do not decrease to excessively
figure 2, no inferences can be made because low levels for at least 2 weeks after
antisera and purified porcine LH used for assay ovariectomy. The relatively slow decrease in
were different. Except for Day 9 (figure 2), LH urinary pregnane excretion as compared to
levels were lower from Days 5 to 14 as decreases in plasma progestin in this study is
compared to Days 3, 4, 21, 28 and 35 after consistent with the relatively high rates of
ovariectomy. These changes suggest that pitu- pregnane excretion observed for several days
itary LH synthesis capacity increased slowly after parturition of sows by Edgerton and Erb
after ovariectomy. The initial increases (Days 1 (1971b).
H O R M O N E S IN B L O O D A N D U R I N E O F SOWS 611
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