y1
Ž l
. The inter-assay coefficients of variation for three control samples were 22 13
y1
. Ž
y1
. Ž
y1
. pmol l
, 6 39 pmol l
and 10 84 pmol l
. The lowest amount of Ž
. oestradiol-17b detectable defined as the intercept of maximal binding y2 SD was 4
pmol l
y1
. Hormone concentrations are expressed in SI units. To convert from pmol l
y1
to pg ml
y1
and from nmol l
y1
to ng ml
y1
the following factors should be used: PGF
2 a
metabolite: 2.8; oestradiol-17b: 3.7 and progesterone: 3.2. 2.5. Analysis of data
Basal concentrations of PGF metabolite were calculated by averaging the values
2 a
obtained on each particular day and removing values higher than two standard devia- tions from the mean value, until the baseline remained unchanged. Concentrations that
exceeded the baseline by more than two standard deviations between days 7 and 12 were defined as peaks. The amount of PGF
released during each peak was estimated by
2 a
calculating the areas under the release curve according to the formula: PGF
release s Ý PGFM q PGFM
r2 = 120 min,
Ž .
Ž .
2 a i
iq120
where i s 0, 120, 240, . . . , min and where PGFM s basal concentration was consid-
i
ered equal to 0. Daily progesterone concentrations were estimated by averaging the concentrations measured in the sample collected at 08:00, 16:00 and 24:00 h for each
Ž .
individual animal. Analysis of variance, using a repeated measures within-SS design, was applied to detect differences in hormone concentrations. In all cases, a least-signifi-
Ž .
cant difference test LSD was used to determine differences between means. Because of variations in the time required to attain peak progesterone concentrations in pregnant
animals, values were normalised against the highest concentration during days 7, 8 and 9. The mean peak value was further compared with mean concentrations attained during
the following days. All statistical analyses were carried out using the StatisticarW,
Ž .
release 4.0, software package Statsoft, USA Statistica for Windows, 1993 . Results are expressed as mean SEM.
3. Results
The progesterone profiles showed that all animals ovulated in response to copulation. Increasing concentrations of progesterone were recorded by day 4 post-mating. Five out
Ž .
of 10 llamas mated with an intact male became pregnant 50 . By day 10–11 after mating, all non-pregnant llamas showed progesterone concentrations close to the detec-
tion limit of the assay. Visual examination of the results for each individual confirmed that a pulsatile
pattern of prostaglandin release occurred during luteolysis in non-pregnant llamas. Fig. Ž .
Ž .
1 a shows the mean SEM plasma progesterone concentrations in non-pregnant animals mated with an intact male and the PGFM secretory pattern in a representative
llama from days 3 to 16 after mating. Although only statistically significant in three animals, slight increases in plasma PGFM were observed as early as day 7 after mating
Ž .
Ž . Fig. 1. Plasma concentrations of progesterone mean, dotted line, SEM, shaded area in non-pregnant a
Ž . Ž
. and pregnant b llamas and a prostaglandin metabolite secretory profile solid line in a representative animal
from days 3 to 16 post-mating. Values identified as significant pulses of PGF metabolite are indicated by
2 a
asterisks. Note the logarithmic scale for the prostaglandin metabolite values.
in most of the animals. PGFM pulses were registered between days 8 and 9 and day 13 after mating in non-pregnant animals. Since no significant differences in the hormonal
secretory patterns were registered between non-pregnant llamas, whether mated with an intact or a vasectomized male, all animals were considered as one group for further
analysis of PGFM profiles. Table 1 shows the characteristics of luteolytic pulses of
Table 1 Ž
. Characterisation of normal luteolytic release of PGF
as determined by PGFM analysis in llamas ns11 .
2 a
Numbers within parentheses in the second column represent the mean number of peaks detected on each particular day. M s 08:00 h, As16:00 h, N s 24:00 h
Days Cumulative
Area under Peak-to-peak
Mean peak Progesterone
y1
Ž .
Ž . Ž
. after
number curve peaks
interval h amplitude
pmol l
y1 y1
Ž .
mating of peaksr
pmol l Ž
. Ž .
pmol l M
A N
animal 7
0.3 2.6
0.1 –
465 7.9
8.3 9.3
Ž .
8 1.4 1.1
49.7 3.6
19.6 748
8.9 7.6
6.5 Ž
. 9
4.3 2.9 358.9
28.6 7.5
1112 4.9
3.3 1.9
Ž .
10 8.2 3.8
520.8 45.7
7.1 1234
1.0 0.7
0.6 Ž
. 11
10.5 2.3 227.2
19.9 9.1
779 0.5
0.7 0.5
Ž .
12 11.6 1.1
23.3 2.1
11.7 566
0.5 0.5
0.5
Table 2 Ž
. Mean SEM plasma progesterone concentrations from days 10 to 16 after mating in pregnant llamas
compared with the mean maximum concentration between days 7 and 9
y1
Ž .
Mean days 7–9 Plasma progesterone concentrations nmol l
Day 10 Day 11
Day 12 Day 13
Day 14 Day 15
Day 16
UU UU
UU UU
UU U
13.00 9.00
8.93 9.26
8.83 8.38
9.66 9.15
1.90 0.72
0.61 0.70
0.63 0.64
0.94 0.83
U
P - 0.05
UU
P - 0.01
Ž .
Ž .
PGFM during luteolysis days 7–12 after mating in non-pregnant animals n s 11 . In Ž
y1
. all cases, the initial pulses were lower in amplitude 525.9 36.7 pmol l
than those Ž
. recorded later
P - 0.01 . The mean peak concentration of the luteolytic pulses was
y1
Ž .
1019 48.5 pmol l . On average, 11.6 range 10–14 peaks per animal were recorded
between days 7 and 12 post-mating. Decreasing concentrations of progesterone were already registered by the afternoon of day 8, when only one or two prostaglandin
metabolite peaks were recorded in each individual animal. Although all animals had reached basal plasma concentrations of progesterone on day 10 after mating, PGFM
peaks were recorded during the following 2 days.
In pregnant animals, plasma concentrations of progesterone remained high until the Ž
Ž .. end of the experiment
Fig. 1 b . Peak plasma progesterone concentrations were attained between days 7 and 9. When the mean progesterone concentrations registered
on those days were compared with the mean concentrations registered from days 10 to Ž
. 16, a significant drop in progesterone was seen after day 9 Table 2 . In connection with
the progesterone decline, PGF pulsatile release was observed in all pregnant animals
2 a
Ž . from days 7 to 15 after mating. Fig. 1 b
shows the PGFM concentrations in a representative llama. The characteristics of prostaglandin release during early pregnancy
Ž .
days 7–12 after mating are shown in Table 3. The overall mean frequency of PGF
2 a
Table 3 Ž
. Characterisation of PGF
release during early pregnancy in llamas as determined by PGFM analysis ns 5 .
2 a
Numbers between parentheses in the second column represent the mean number of peaks detected on each particular day. M s 08:00 h, As16:00 h, N s 24:00 h
Days Cumulative
Area under Peak-to-peak
Mean peak Progesterone
y1
Ž .
Ž . Ž
. after
number curve peaks
interval h amplitude
nmol l
y1 y1
Ž .
mating of peaksr
pmol l Ž
. Ž .
pmol l M
A N
animal 7
0.4 11.1
0.1 –
494 7.6
6.2 8.7
Ž .
8 1.0 0.6
6.0 3.6
– 443
9.1 11.3
8.2 Ž
. 9
1.4 0.6 11.0
28.6 28.0
488 10.0
8.0 7.3
Ž .
10 2.2 0.8
69.3 45.7
35.3 676
10.0 9.0
8.0 Ž
. 11
2.4 0.2 7.3
19.9 38.0
388 8.7
9.8 8.4
Ž .
12 3.0 0.6
28.8 2.1
99.0 547
8.0 10.4
9.4
Ž .
Ž . Fig. 2. Plasma concentrations of oestradiol-17b meanSEM, shaded area in non-pregnant a and pregnant
Ž . b llamas from days 3 to 16 post-mating.
metabolite peaks detected in pregnant animals was 0.64 peaksranimalrday, while the mean peak amplitude was 503.8 49.1 pmol l
y1
. Oestradiol-17b plasma concentrations increased slowly from days 3 to 8 after mating
in all lamas. Thereafter, concentrations sharply increased in non-pregnant llamas, until Ž
y1
. peak concentrations 30.9 3.8 pmol l
were attained on day 15. Conversely, a slow oestradiol-17b rise was recorded in pregnant animals in which the highest concentra-
Ž
y1
. Ž
Ž . Ž .
tions 11.2 4.9 pmol l were observed 12 days after mating Fig. 2 a and b
. respectively .
Ž .
Fig. 3. Plasma concentrations of progesterone mean, dotted line, SEM, shaded area in non-pregnant llamas Ž .
Ž .
mated with a vasectomized male a and in non-pregnant llamas treated with FM black bar four times a day Ž .
Ž .
b , and a prostaglandin metabolite secretory profile solid line in a representative animal from days 5 to 12 post-mating. Values identified as significant pulses of PGF
metabolite are indicated by asterisks.
2 a
Prostaglandin metabolite concentrations decreased rapidly in all animals after the first administration of FM from about 450 pmol l
y1
to concentrations of around 180 pmol
y1
Ž .
l P - 0.01 . Concentrations of the metabolite remained low throughout the first 2
Ž .
days of FM treatment days 6 and 7 post-mating . Thereafter, pulsatile release of PGF
2 a
started, and concentrations similar to those recorded during the first 2 days of treatment were recorded between peaks.
Fig. 3 shows the changes in hormone concentrations in non-pregnant llamas mated Ž .
Ž . with a vasectomized male during normal luteolysis a and during FM treatment b . In
Ž .
llamas treated with FM, the cumulative number of peaksranimal were 1 day 8 , 2.2 Ž
. Ž
. Ž
. Ž
. day 9 , 4.4 day 10 , 5.8 day 11 and 6 day 12 . The mean peak prostaglandin
y1
Ž .
y1
Ž .
y1
metabolite amplitudes were 261 pmol l day 8 , 547 pmol l
day 9 , 561 pmol l Ž
.
y1
Ž .
y1
Ž .
day 10 , 993 pmol l day 11 and 864 pmol l
day 12 . Increasing concentrations of progesterone were recorded from days 5 to 7 post-mating in both groups. Thereafter,
progesterone concentrations declined and were close to the detection limit of the assay by day 9 post-copulation in untreated animals. Progesterone concentrations remained
high until day 8 post-mating, after which, they started to decrease until basal concentra- tions were attained by day 10 or 11 post-mating.
4. Discussion
