PREGNANCY AND OVULATION DETECTION IN BISON (BISON BISON) ASSESSED BY MEANS OF URINARY AND FECAL STEROIDS Author(s): Jay F. Kirkpatrick, K. Bancroft, and V. Kincy Source: Journal of Wildlife Diseases, 28(4):590-597. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-28.4.590 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-28.4.590
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Journal
PREGNANCY
AND
ASSESSED
OVULATION
BY MEANS
DETECTION
OF URINARY
of
Wildlife
IN BISON
AND
FECAL
Sexually
lation by diagnosis
mature
means of urinary was determined
bison (Bison bison) steroid metabolites among 18 bison cows,
28(4), Disease
1992, pp. 590-597 Association 1992
(BISON
BISON)
STEROIDS
Jay F. Kirkpatrick,’ K. Bancroft,2 and V. Kincy2 1 Deaconess Research Institute, 1500 Poly, Billings, Montana, 59102, USA 2Department of Biological Sciences, Eastern Montana College, Billings, Montana
ABSTRACT:
Diseases, © Wildlife
59101,
cows were tested and fecal steroids.
USA
for both pregnancy The accuracy of
and ovupregnancy
in approximately the third month of gestation, by means of urinary pregnanedio!-3#{244}-glucuronide (PdG), urinary estrone conjugates (E,C), and fecal total estrogens (TE). Urinary PdG was 100% accurate, urinary E1C was 89% accurate, and fecal TE was 100% accurate in predicting pregnancy. Fecal progesterone (P4) and TE, as well as urinary EC and PdG concentrations all increased from conception (August) through January, but significant differences were not apparent until November. During the rutting season ovulation was detected by increases in either urinary PdG or fecal P4 concentrations. Both pregnancy and ovulation were detected in uncaptured bison with reasonable accuracy by means of urinary and fecal steroids or their metabolites. Key words: Bison, Bison bison, ovulation, pregnancy, reproduction, steroids, urine, feces.
INTRODUCTION
detection total and
Until recently the study of reproductive physiology in large free-roaming wildlife has required immobilization on capture of animals to measure blood. During the of pregnancy and ovarian function have been based
endocrine past decade, non-invasive in captive exotic on measurement
nary 1983; from
steroid Lasley, certain
metabolites 1985). Urine free-roaming
used
in
study
the
levels diagnosis study
of
et al., 1991a). Very little
in of
species of uri-
means olites, by
Kirk-
and
cycles
Kirkpatrick,
ican bison Kirkpatrick While
in 1991)
feral and
horses North
means
study stand
(Lasley Amer-
regulation
(Bison bison) (Kincy et al., 1990; et a!., 1991b). these techniques are successful,
success pressures.
collection of urine, even in snow, can be time-consuming. Use of feces for similar studies has such advantages as easily observable elimination by the animal and relatively strategy
means
of
of fecal
et a!., 1990b) (Kirkpatrick about
the
repro-
fecal steroid could be
urinary
PdG hypotheses of fecal urinary
and
metabdetected feca!
pro-
tested estrogens estrogen
were and and
progesterone metabolites differed significantly between pregnant and non-pregnant animals, and (b) ovulation could be detected by identification of a luteal phase pattern of excretion of fecal progesterone and urinary progesterone metabolites. Our
gesterone metabolites (iPdG). Collection of serial urine samples has permitted the characterization of conceptive and nonconceptive
by
of free-roaming bison. this study were to decould be detected by
of urinary and and if ovulation
gesterone (P4). The (a) concentrations progesterone and
patrick et a!. (1988, 1990, a, b) diagnosed pregnancy in uncaptured feral horses (Equus caballus) by measuring urinary estrone conjugates (E1C) or non-specific pro-
horses
is known
ductive physiology The objectives of termine if pregnancy
(Loskutoff et a!., can be collected wildlife and
reproduction.
in feral
estrogens (Kirkpatrick fecal steroid conjugates
was based on the mechanisms and that
the
the desire to of reproductive
changes
accompany
MATERIALS
underself-
in reproductive environmental
AND METHODS
Animals in this study were part of a herd of approximately 80 bison inhabiting a 200 ha range in northern Wyoming (44#{176}44’N, 109#{176}2’W). The vegetation included predominantly mixed-grass prairie and has been described by Knight et a!.
easy location of the excrement. This has led to successful pregnancy 590
KIRKPATRICK
(1987).
Cows
ranged
in age
from
3 to 17 yr.
Six
sexually mature bulls were present among the herd at all times. Water was available from natural sources ad libi’tum and feed was natural grass. Individuals were identified by numbered ear tags. Experiment 1: During November 1988, bison were observed from a distance of 50 to 100 m; urine samples were collected from 18 cows. Urine was aspirated from pools on the ground or extracted from freshly soaked earth as described by Kirkpatrick et a!. (1988). Samples were kept on ice during the day of collection and stoned at -7 C without preservatives until tested 2 to 3 mo later. Fecal samples were stored in plastic bags at -7 C until tested. Urine samples were diluted 1:2 with distilled water (dH2O); 20 l of the diluted sample were analyzed for pregnanediol-3#{246}-glucuronide (PdG) and E,C. The enzyme immunoassays for PdG and E1C have been described previously (Kirkpatrick et al., 1990a; Munro et al., 1991) and the PdG assay has been validated in bison (Kincy et al., 1990; Kirkpatrick et al., 1991b). To account for differences in urine concentrations, urine samples were diluted from 1:50 to 1:100 with dH2O and analyzed for creatinine (Cr) concentrations by the microcolorimetnic method of Taussky (1954). Pregnanediol-3-#{244}-glucuronide and EC values were calibrated to Cr values and reported as ng/mg Cr. Wet fecal samples were weighed and 0.5 g samples were extracted in ethyl acetate/hexane (3:2, V/V) and analyzed for total estrogens by radioimmunoassay (Kirkpatrick et al., 1990b). The cross-reactivity of the anti-estrogen antibody with 17#{244}-estradiol was 11% (Kirkpatrick et al., 1990b). Approximately 5,000 counts per minute (cpm) of 3H-17 estradiol was added to each fecal sample before extraction in order to estimate recovery of estrogens. Total estrogens were reported as ng/g wet feces. In January 1989, the bison were confined in chutes and tested for pregnancy by rectal palpation; calves were counted during April, May and June. Based on the dates of partunition, 12 of the conceptions had occurred between 15 July and 10 August 1988.
Experiment 2: Fecal samples were collected from six bison cows in September, November 1989 and January, 1990. Weighed samples were extracted with dH2O, the residue dried and weighed, 0.25 g was rehydrated and extracted with diethyl ether, and redissolved in 95% ethanol by the methods of Desaulniers et al. (1989). Approximately 10,000 cpm of ‘I-progesterone was added to each fecal sample before extraction in order to estimate recovery of P4. Twenty
ET AL-PREGNANCY
,l of the dHp,O
AND OVULATION
extract
were
IN BISON
analyzed
for PdG
and EC. The ethanol extracts were 1:100 to 1:1,000 in dH2O and 20 il were for total estrogens by radioimmunoassay
patrick et al., 1990b), immunoassay (Munro
and and
591
for P4 by Stabenfeldt,
diluted
assayed (Kirk-
enzyme
1984). given as ng/g dry feces. Mean for September 1989, November 1989, and January 1990 were tested for significant differences with Student’s t-test (Freedman et al., 1978). Pregnancy was confirmed by rectal palpation in January 1990, and calf counts
All values concentrations
are
were made in April, May and June, 1990. Dates of partunition indicated that five of the six conceptions occurred between 25 July and 15 August.
Experiment 3: Urine and fecal samples were collected approximately every 2 to 4 days during the rutting season from five sexually mature cow bison between 31 July and 31 August 1989. Urine samples were collected, stored, and analyzed for PdG as described in experiment 1. On some days both urine and fecal samples were collected from the same cow but on other days only
one
samples yl ether as
type
were and
described
of
sample
was
collected.
weighed and extracted the residue redissolved by
Desaulnier
et
Fecal
with diethin ethanol (1989). The
al.
ethanol extracts were diluted 1:100 in dH2O and 20 zl were analyzed for P4 (Munro and Stabenfeldt, 1984). The patterns of urinary PdG and fecal P4 excretion were recorded for each bison cow and compared by regression analysis; the correlation coefficient for the two hormones was determined (Freedman et al., 1978). RESULTS
Experiment 3H-17f3 (±6.45)%. nant
1:
Mean
estnadiol Fourteen
during
January
by rectal palpation, calves during 1989. gen analysis was pregnancy, with lowest value in
(±SE)
recovery
from feces of 18 cows 1989 and The
was were
as determined all 14 delivered total fecal estro-
the most accurate 1.09 ng/g feces pregnant cows
ng/g being the highest value pregnant cow (Table 1). Using feces
as the
total accurate cy,
minimum
fecal estrogen as a diagnostic
of 62.18 preg-
value analysis test for
for
test for being the and 0.20 in
a 1.0
nonng/g
pregnanwas 100% pregnancy.
Urinary PdG analysis was equally accurate. The lowest urinary PdG value for pregnant cows was 54.7 ng/mg Cr while 9.9 ng/mg Cr was the highest value for a
592
JOURNAL
TABLE
1.
OF WILDLIFE
Urinary
concentrations
Bison
DISEASES,
VOL. 28, NO. 4, OCTOBER
(PdG),
pregnanediol-3#{244}-glucuronide
for
reproductive
pregnant
bison
at
group
Pregnant
(n
three
urinary
gestation,
PdG (ng/mg Cr)
estrone and
conjugates
nonpregnant
EC (ng/mg
(EC),
and
54.7
Mean SE
113.30 18.57
Total
(n
9.4
to 302.9
to 83.5
1.09
26.74 5.65
4)
=
3.6 6.82
5.62
0.16
SE
1.55
2.03
0.02
cow.
between
urinary
PdG
The
total was
to 9.9
2.7
correlation
fecal
r
0.70
=
coef-
estrogens (P
and 0.001).
10.0 ng/mg Cr minimum PdG value for pregnancy,
as the uri-
nary PdG analysis was 100% accurate in predicting pregnancy. The urinary E1C analysis was less accurate in predicting pregnancy. for pregnant the
(P
The lowest cows was
highest
pregnant correlation estrogens =
E1C
nary
urinary E1C value 9.4 ng/mg Cr and
concentration
0.049).
Using E,C
E1C
10
value
analysis
ng/mg for
was
all rose between approximately
September 0.001) Both
E1C
and
concentrations tion period the estrous Asdell, bonasus)
had cycle
Urinary
November,
PdG
but
did Janrose
and
European bison and Raczynski,
fecal collecwith taurus) (Bison 1967).
from feces P4 concen-
ranged to 3,000
beng/g
feces and reached a peak of 5,000 to 10,000 ng/g midway through the luteal phase (Fig. 2). These values were similar to those in domestic cows (Desaulniers et al. 1989). Basal urinary PdG ing ovulation ranged mg Cr and reached
concentrations precedbetween 5 to 100 ng/ a peak of 350 to 1,000
during
phase (Fig. 2). The between urinary the five bison cows
the
August
rut
ranged
a low of r = 0.310 to a high of r however, in all five cases the luteal
pattern
of P4 secretion
hormones.
Four
was
of the
evident
five
=
for both
bison
demon-
strated classic estrous behavior which was coincidental with both PdG and P4 nadins immediately preceding luteal phase elevation. Four of the five bison had a second estnous dates
of
cycle during parturition
those second conception.
Septem-
during the 32-day a pattern consistent of the cow (Bos
1964) an the (Krasinski
ovulation 2,000
from 0.922;
between November and fecal P4 and total estrogens
3:
preceding approximately
monitored
4.5 mo postconE1C and PdG did (P < 0.05) be-
Experiment
trations tween
the
and
to 0.2
Mean (±SE) recovery of 19-P4 was 61.8 (±5.3)%. Basal Fecal
uri-
as
and PdG concentrations
(P 35 days postconception) detection of pregnancy in the Penisso-
(B)
through
urinary
of
January
Collection
pregnanediol-3#{246}-glucuronide
approximately
180 days
of pregnancy.
dactyla and particularly the Equidae (Kassam and Lasley, 1981; Kasman et al., 1985, 1986, 1988; Czekala et al., 1990), but these early
steroid detection
Bovidae. metabolize
metabolites are of pregnancy
This is probably plasma estrogens
of conjugated
point where predictors
of
useful among
because to other
compounds.
to 4 mo of pregnancy, trations have increased
not
for the
bovids forms
However, estrogen in bovids
by
estrone conjugates are pregnancy, despite
useful repre-
senting only a minor urinary metabolite. This is corroborated by the dramatic crease in fecal estrogens demonstrated Safer-Hermann
et a!.
(1987)
3
concento the
in cows,
inby red
594
JOURNAL OF WILDLIFE DISEASES, VOL. 28, NO. 4, OCTOBER 1992
6000
400
(i
-5000 300
E
E -4
0I
C
#{163}
#{163} 0
o
0.
0 .5
0.
a
0.
a
IL.
.,
0
200
a. 3
-
U
100
a
2000
30
Day
+10
20
Day
Coll.ctlon
of
a
U
a
of
0
30
CoIl.ctlon
7000
500-
c5
C,
6000
400-
E
.
5000 C
C
300 -4000
0 200’
0
a.
0
0
0.
:
3000
a U .
a U.
U.
2000
100’
,.
0
.
10
20
Day
of
10
40
30
Day
ColI.ctlon
20 of
CoIl.ctlon
1000
PdG(ngmgCr)
-=
FscalPo(ngi9)
4-
C)
E C C
0
0.
C .5 0.
S U S
U.
a C
Day
2.
FIGURE
excretion
Patterns
during
of
the luteal
urinary
phase
of
ColI.ctlon
pregnanediol-3#{244}-glucuronide
for five bison
cows.
(PdG)
Arrows
indicate
and
fecal
the days
progesterone
of observed
(Fecal
behavioral
Po)
estrus.
buffalo (Syncerus caffer nanus) and yak (Bos mutus). The primary fecal estrogen during pregnancy in cows, after 90 days postconception, is 17#{244}-estradiol (Most! et a!., 1984), and the elevation of estrogens is great enough that an estrogen radioimmunoassay with only an 11 % cross-reac-
sitive enough for pregnancy detection. The assay in this study, however, was used to measure total estrogens, and the presence of 17#{246}-estradiol in pregnant bison is only assumed. Use estrogen
of
tivity
nancy
detection
with
this
particular
metabolite
is sen-
urinary PdG, concentrations in bison
E1C, on fecal for accurate was
most
total preg-
reliable
KIRKPATRICK
after 90 days postconception. The of increase in fecal total estrogens nary E1C in plasma
in the total
gens seen (Desaulniers
in
The small imals in this the
full
bison is similar estrogens and
the pregnant et al., 1989). number study
range
to changes fecal estro-
domestic
cow
of non-pregnant prevented analysis
of
values
measured. Perhaps with of non-pregnant animals, mone curacy crease.
pattern and un-
values would of pregnancy
be
for
the
steroids
a larger the range
greater diagnosis
anof
number of hor-
and the might
acde-
Ovulation and formation of the corpus luteum could not be confirmed by rectal palpation or ultrasound in bison of this study. detected table
While by
presence of a fetus could be rectal palpation, the intrac-
nature
of the
animals
did
not
permit
ET AL-PREGNANCY
AND OVULA1]ON
traction. Conjugated are excreted primarily after being resorbed vascular bed; however,
IN BISON
steroid metabolites by way of the urine, from the gut into the a small proportion
of the conjugates remain trapped gut and are excreted in the feces and Kirkpatrick, 1991). These fecal conjugates the ether
remain extraction on
water used
based
niers this
et a!. (1989), who successfully method to the fecal analysis
recent
muskoxen P4 could
not
in the (Lasley steroid
soluble. Finally, for progesterone
was
cows and Tnitiated
the
595
work
of
Desaulapplied of P4 in
(Ovibus moschatus). be used for deter-
mination of P4 recovery because large amounts of plant pigments also are removed with the ether extraction, and the interference of the spectrophotometny is too great for liquid scintillation counting. Validation
of
urinary
and
fecal
steroid
the sensitive palpation techniques required for detection of corpora lutea. Ultrasound was not available. Despite the in-
analysis in large intractable ficult and cannot always procedures used with
ability these
(Lasley and Kirkpatrick, 1991). Validation of these methods rests on several assumptions. First, it has previously been dem-
to confirm direct methods,
ovulation by the pattern
either of of P4 se-
cretion was indicative of ovulation. There can be at least three causes for increases in P4, including ovulation, formation of a persistent
corpus
luteum,
on extra-ovarian
(adrenal) formation
P4. However, only ovulation and of the corpus luteum result in
bison
the P4 pattern seen in these bison. There was an evident 2 to 3 day lag in excretion of fecal P4 compared to the excretion of urinary PdG. Despite the coefficient between urinary P4 in some of the animals, pattern was clearly evident indicator
low correlation PdG and fecal the lutea! phase and a reliable
of ovulation.
Several were used
different for the
extraction recovery of
onstrated between
methods steroids or
Second, matography validate mary
the
between
(Kirkpatrick
et a!., validation of the
traction
measured.
organic
solvent
ex-
bison
This (1975),
primary cow also
and the PdG have been
physiological ovulation and precise nature
than
in
1990c). Fehen the
trogens and diagnosis of pregnancy in feral horses (Kirkpatrick et a!., 1990b). Extraction of E1C and PdG required water exrather
plasma
in
therefore, PdG and 2) imply
and
fecal
P4.
performance liquid chropreviously has been used to presence of PdG as the pri-
P4 metabolite
parallelism
1991b); urinary here (Fig.
between high
strated that the domestic
timate measure
the es-
et a!.,
patrick et a!., the work of
covery previous
based on of fecal
(Kirkpatrick
a correlation
ples ously
of free estrogens was successful extraction
that there is a strong correlation plasma P4 and urinary PdG
the correlations between fecal P4 demonstrated
their conjugated metabolites for the three experiments. The ethyl acetate/hexane extraction used in experiment 1 for the re-
animals is diffollow the same captive animals
(Kirk-
P4 metabolite in was PdG. Third,
serially standard demonstrated 1991b).
urine
is supported by who demon-
diluted curve Finally,
of these techniques accuracy in predicting events in question, pregnancy, rather of the metabolites
sampreviin bison the
ulis a the
namely than the being
596
JOURNAL OF WiLDLIFE DISEASES, VOL. 28, NO. 4, OCTOBER 1992
ACKNOWLEDGMENTS The
authors
gistical
gratefully
support
of the
Bar-X
study
was
acknowledge
provided
Ranch,
by Mrs.
in Powell,
funded
in part
by
tional Science Foundation 01 and DCB-8922800.
1964.
CZEKALA,
N.
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This
Xi and
Na-
H.
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