ISRAEL JOURNAL OF
VETERINARY MEDICINE
ESTRADIOL-17β AND PROGESTERONE PROFILES OF FEMALE
CAMELS AT DIFFERENT REPRODUCTIVE STAGES
Deen, A., Vyas, S., Sahani, M.S., Saharan, P.*, Sevta, I.*, and Chabra, S.*
National Research centre on camel, Jorbeer, P.B. No. 07, Bikaner
RAJASTHAN, INDIA
Telephone 2234303 151 0091 Telefax 2231213 151 0091 E-mail
:aminudeen@scientist.com
Abstract
The study was conducted on 56 female camels for E2 and P4 profiles at
different reproductive stages, viz. 35 bred females
(Group A) monitored after breeding once daily for 0-30 days were divided
into 2 groups of pregnant (n=13) and nonpregnant
(n=22) based on P4 profiles, another pregnant group (Group B) (n=8) was
monitored at weekly intervals from 23rd weeks to the end of gestation;
periparturient camels (Group C) were monitored at 6h intervals, while
nonpregnant females (n=7) (Group D) with growing and mature follicles
were monitored for E2 profiles only and the final group (Group E) (n=6) of
nonpregnant females was monitored for E2 profiles before and after
mating. The average P4 concentrations in pregnant and nonpregnant
females of group A were similar from days 0-10 after mating. They declined
from day 11 onward in nonpregnant females, but continued to increase in
pregnant animals (P<0.01). The average daily E2 profiles were found to be
low or basal in both non-pregnant (1.32 to 8.74 pg/ml) and pregnant
females (0.69 to 8.24 pg/ml). The average concentration of P4 in group B
was relatively higher (5.87 to 12.07 ng/ml) between 23rd to 32nd weeks of
gestation than at later stages (2.88 to 5.09 ng/ml). The average
concentration of P4 recorded in periparturient female camels of Group C
was around 4.0-4.5 ng/ml at 55-31 hrs prior to parturition and declined
slightly to measure 3 ng/ml at parturition. A further decline in P4
concentration to 1.6 ng/ml occurred after expulsion of the fetus. The
average concentration of E2 was low up to 38th weeks of gestation. It
started to increase slowly and steadily after the 39th week and measured
more than 50, 100, 250, 300 and 375 pg/ml at the 42nd, 45th, 47th, 49th
and 52nd weeks of gestation, respectively. It declined in periparturient
females to 92.2-243 pg/ml at 1-55 hrs before calving. It further declined
sharply to 23.3, 5.6 and 6.6 pg/ml at 5, 11 and 17 hrs after calving.
E2 profiles of nonpregnant females of group D (n=7) with mature sized
ovarian follicles monitored at 30 minute intervals for 2 hrs daily for 15–20
days (for E2 profiles only) revealed mostly basal levels with a few
intermittent peaks, indicating the pulsatile nature its secretion One group of
nonpregnant females, Group E (n=6) with mature follicles monitored for E2
profiles only, one day prior to and immediately after mating showed that E2
profiles ar these times did not differ.
Key words: Camel; Radioimmunoassay; Estradiol.; Progesterone; Pulsatile
Introduction
Estrogen and progesterone are 2 important female sex steroid hormones,
whose rhythmical secretion determines the reproductive behavior, oestrus,
ovulation, pregnancy and parturition. The pattern of secretion of these
hormones has been well-documented in cattle, buffalo, sheep, goat, mare and
pig but is less well understood in the camel. Camel is different in the sense
that ovulation is of an induced type rather than the spontaneous type in most
species (1). The ovulation and luteal phases are induced only in bred females,
while unbred females remain unovulated and display follicular phase only (2).
Rhythmic secretion of these sex steroids has a definite correlation with sexual
behavior and receptivity of the male by females in other species of livestock.
In camelids the periods of estrous and non-receptivity do not necessarily
coincide with ovarian status and levels of estradiol-17β and progesterone.
*Students of Bio technology Some alpacas and llamas may refuse to mate,
regardless of ovarian status (2). The duration of estrus or receptivity and the
period of non-receptivity to mating in camelids are unpredictable and highly
variable. The relationships between the mating behavior of female alpacas,
llamas and dromedary camels together with the associated ovarian follicular
dynamics including secretion and levels of ovarian steroid hormones, have
not been established. Estrogen in camels has been reported to be highly
variable and seemingly impossible or difficult to interpret (3). It is speculated
but not definitely confirmed that for camelids, as for other species, the
concentration of estradiol-17β is critical for triggering the ovulatory surge of
LH (2). It is also speculated that immature and small growing follicles secrete
estrogens, but not in amounts sufficient to induce the ovulatory surge of LH.
Matings that occur during the final stages of follicular growth or when the
follicle is mature induce the ovulatory surge of LH, but the nature of the preovulatory estradiol surge and its occurrence in relation to mating is not known
in camels. Corpus luteum in non-pregnant females is short-lived. The
precipitous fall in P4 concentration in plasma does not occur at the time of
parturition (4). Therefore the present experiment was undertaken to study the
profiles of estradiol (E2) and progesterone (P4) in relation to different
reproductive stages, follicular status, mating, pregnancy and periparturient
stages and become fully acquainted with reproductive events of camels.
Materials and methods
Animals:
The study was conducted on 56 female dromedary camels of Bikaneri breed
of 7-10 years of age that were maintained under semi-arid climatic, and semiintensive husbandry conditions in the western part of India, the Renn of
Kucchh.
The following groups were included: 35 female camels (group A), which were
bred with males and monitored for E2 and P4 profiles once daily from 0-30
days (Day 0=day of mating). This group was then divided into pregnant
(n=13) and non-pregnant (n=22) subgroups based on their P4 profiles and
recto-genital palpation;
Group B of 8 pregnant females was monitored at weekly intervals from 23rd
week to the end of gestation and at 6hr intervals at the periparturient period
(group C).
Group D of 7 female camels, which had growing and mature follicles were
monitored only for E2 profiles at half-hour intervals for 2hrs daily over 15-20
days to follow the secretion of this hormone.
Group E of 6 female camels, which had mature follicles were monitored for E2
profiles at half -hour intervals for 6hrs before as well as after mating to
evaluate if it had any effect on secretion of the hormone. 2.2.
Quantification of E2 and P4: The quantification of E2 and P4 were carried
out by radioimmunoassay (RIA) using anti-estradiol and antiprogesterone
serum and tritiated estradiol and progesterone, as follows: Anti-estradiol and
anti-progesterone sera were obtained from Professor Niswender, Colorado
State University, USA. Lyophilized antiserum was reconstituted with Millipore
filtered water to 1 ml of antiserum. The reconstituted antiserum was aliquoted
to 10-20 μl and kept at –40°C till used.
Radiolabeled estradiol and progesterone: 2,4,6,7,16,17-3H estradiol and
1,2,6,7,16,17 3H progesterone were purchased from Amersham Bio-Sciences
Limited, UK. Antibody titer curves were run to determine the titer used in the
assay. Stock calibrators were prepared in alcohol and working calibrators in
PBS. Incubation of antibody, sample and tritiated tracer at 4OC for overnight
was followed by separation of loose and bound fractions of tracer by the
charcoal dextran method. The bound fraction was decanted into a scintillation
vial followed by addition of scintillation fluid. Counting for β-disintegrations
was accomplished in a Beckman liquid Scintillation Counter
LS 6500. Calibrators were used to quantify the hormones in the samples.
Assay validation
Sensitivity: Progesterone: 0.1ng/ml; Estradiol 17β: 3.0pg/ml.
Specificity: The antibody used in the immunoassay was highly specific for
estradiol and progesterone. Extremely low crossreactivities were obtained
against naturally occurring steroids.
Intra-assay precision: Samples were assayed 10 times in the same series.
The coefficients of variation were found below or equal to 5.8% and 4.9% for
oestradiol and progesterone, respectively. Inter-assay precision: Samples
were assayed in duplicate in 18 different series. Coefficients of variation were
found below or equal to 9% of plasma sample.
Accuracy:
a) Dilution test: High-concentration samples were serially diluted in a sample
with a low concentrations of progesterone and estradiol. The recovery
percentages obtained were between 87% and 115%.
b) Recovery test: Low concentration samples were spiked with known
quantities of progesterone and estradiol. The recovery percentages obtained
were between 85% and 110%.
Statistical analysis:
E2 and P4 profiles of pregnant and non-pregnant females from day 0-day 30
were compared by SPSS (5)Harvey, 1987). Means of the 2 groups on
different days were compared by t-test.
Results:
Eestradiol-17β (E2) and progesterone (P4) profiles of camels were studied at
different stages of reproduction. The results are presented in Table 1 and
Fig(s) 1, 2, 3, 4, 5 & 6.
Progesterone profiles:
Bred camels for 1st month of gestation: The average P4 concentration in
pregnant and non-pregnant female camels measured around 0.5 ng/ml of
plasma from day 0-3, increased slowly thereafter and measured above 1
ng/ml on day 6. It continued to increase further in both the groups till day 11,
after which non-pregnant females exhibited a significant decline while
pregnant ones showed an increasing trend to maintain a plateau around 3 4.14 ng/ml on subsequent days (Figs. 1 & 2).
Length of luteal phase: Table 1 shows the related data, which indicate that
length of luteal phase varied from 0 to 11 days. Stage of luteal phase in
relation to mating varied from days 2-4 to 2-12 and the peak values varied
from 1.4 to 4.5.
Pregnant camels at latter half of gestation (group B): The average
concentration of P4 was relatively higher (5.87 to 12.07 ng/ml) between 23rd
to 32nd weeks of gestation than at later stages (2.88 to 5.09 ng/ml) (Fig. 3)
Periparturient female camels (group C): The average concentration of P4
measured 4.01±0.35, 3.98±0.43 at 55 and 31 hrs prior to parturition,
respectively, which declined only slightly to be around 3.13±0.40 ng/ml at the
time of parturition. Further decline in P4 concentration occurred after
expulsion of fetus measured around 1.65±0.11 ng/ml (Fig. 4).
Estrogen profiles: Pregnant and non-pregnant camels for 1st month of
gestation (group A): The average daily E2 profiles for pregnant and nonpregnant females varied from 0.69 to 8.24 pg/ml and 1.32 to 8.74 pg/ml,
respectively and did not differ significantly between two groups (Figs 1-2).
Pregnant camels for latter half of gestation (group B): The average
concentration of E2 was low (16.18±3.69) up to 38th weeks of gestation.
Thereafter, the concentration of E2 started to increase slowly and measured
72.5±21.68, 116.32±24.76, 227.5±56.25, 308.57±86.04 and 375±110.26
pg/ml at 42nd, 45th, 47th, 49th and 52nd weeks of gestation, respectively
(Fig. 3).
Periparturient female camels (group C): The E2 concentration started
declining in periparturient females and fluctuated between 92.2±29.5 to 243
pg/ml at 1-55 hr before calving. The concentration of E2 declined sharply after
calving and measured 23.3±23.2, 5.6±3.3 and 6.6±4.9 pg/ml at 5, 11 and 17
hrs respectively, after calving (Fig. 4).
E2 profiles of 7 non-pregnant camels of group D with mature ovarian follicles
monitored for 15-20 days for 2 hrs daily at 30 minutes intervals are presented
in Fig. 5. It depicts that the concentrations of E2 in these females were basal
mostly with several intermittent peaks indicating that its secretion from mature
follicles might be pulsatile.
E2 profiles of another 6 non-pregnant female camels (group E) with maturesized ovarian follicles were monitored; one day prior and immediately after
mating for 6 hrs at 30 min intervals, and results depicted in Fig. 6 show that
the E2 profiles did not differ prior to and after mating and mating does not
appear apparently to have a stimulating effect to induce E2 surge similar to
that described as the pre-ovulatory E2 surge in other species. Any such
surge, if it occurs in camelids could not be characterized with these efforts.
Fig.5: Estradiol-17β profiles of non-pregnant camels with follicular activity in ovaries
Fig.6: Estradiol-17β Profiles of 6 female camels bearing mature ovarian follicle –1
day and after mating
Table 1: Length of luteal phase, its relation to the mating and peak P4 values
recorded in female nonpregnant Camel
Discussion:
E2 and P4 are the two important female sex steroid hormones,rhythmical
secretions of which regulate the reproduction in females.
E2 profiles: Cycling camels: Low (0-9 pg/ml) daily E2 profiles observed in
the present study resembled values reported by Young (6) of 12.11± 4.44
pg/ml on day 0 and 8.39 ± 1.92 pg/ml on day 4 after mating. E2 concentration
had a wave-like pattern in the vicuna (7), and llama (8). E2 concentrations
varied between 12-62.8 pmol/lt in vicuna, while peak concentrations of
46.9±3.3 pmol/ lt attained 12 days after initiation of follicle growth in the llama.
Similar values varying from 9-110 pg/ml with a peak values of 74 pg/ml
maintained for an average of 2.9 days were reported in the dromedary (9).
Such high values were not recorded in the present study. Tibary and
Anouassi (3) cited several reports indicating that peak values persist for 4 to
15 days or for the entire breeding season and that these peaks are repeated
at intervals of 13 days. These peaks were not observed in the present study.
In fact E2 concentrations monitored once daily in present study has shown
that majority of samples had very low, basal or undetectable concentration of
this hormone despite the camels having mature and growing follicles,
whereas pulsatile E2 secretion was found in multiple sampling in unmated
females with follicles. Contrary to the general assumption that dramatic
changes occur in E2 and P4 hormone concentrations during
the estrous cycle after a longer interval and subtle variations are expected to
be very small. Pulsatile secretion of pituitary hormones is well known.
Similarly, pulsatile secretion and release of testosterone and glucocorticoids
have been reported (10) and pulsatile secretion of P4 from corpus luteum was
reported in cattle. In contrast, reports of pulsatile secretion of E2 from follicles
as observed in present study were not found in the literature on camel
reproduction.
Effect of mating: Mating did not induce a pre-ovulatory E2 surge. It appears
that the camel does not have a E2 surge as reported in other species. The
occurrence of ovulation is thus questionable. Karsch et al. (11) observed in
sheep that estradiol contributes to LH peak but it is not necessary that it
should have a rising trend like a surge. Low levels of E2 might be sufficient to
induce LH peak essential for ovulation also in the camel.
Pregnant animals: A significant rise in E2 concentraion was observed
around the 39th week of gestation, which continued to rise till the last week of
gestation. This finding resembles other reports (9, 12, 13, 14), Periparturient
period: E2 concentration declined rapidly after the onset of labour and basal
levels were attained soon after the expulsion of the fetus. These observations
are similar to those reported for camels by others (9, 13 ,14).
Progesterone: Pregnant: In 13 female camels which conceived, the average
P4 concentration rose above 1ng/ml on day 6 followed by continued rise on
subsequent days. Initial rise in P4 concentration above 1 ng/ml varied widely
from day 2 to day 15, which indicated that ovulation and development of
corpus luteum vary greatly between individual in relation to mating. The
values observed in present study resembled all those reported previously in
camel studies (13, 14).
Non-pregnant: The average P4 concentration from day 0 to day 10 rose in
similar fashion to those of pregnant. The magnitude of P4 was less than of
pregnant animals but the difference was non-significant on these days. On
day 11 onward, the average P4 concentration declined and was significantly
lower than pregnant females. These findings resembled to those reported by
others (15). The length of the luteal phase was relatively shorter than of other
livestock species. Of interest in the present study was that the length of the
luteal phase was not only shorter than that of other species but it varied
greatly among the females from 2 to 11 days. This resembled to the findings
of Skidmore et al (16), who reported a life span of 8.5±0.5 days for the corpus
luteum (7-12 days).
Latter half of gestation: P4 values observed in the present study resembled
those Periparturient animals:- The concentration of P4 did not decline to the
extent observed in cattle, sheep and goat where the P4 declines to an
undetectable level before parturition and where infusion of progesterone will
delay parturition. The present study findings are similar to those of Zhao and
Chen (4), but differ from those of Tibary and Annouassi (3). The occurrence of
parturition at high peripartal concentrations of P4 in mares and camels needs
more investigation
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