treatments at different stages of the oestrous cycle on follicle growth have been Ž
. examined in ewes Leyva et al., 1998 . The results were equivocal due to the lack of
continuous data on the growth of ovulatory follicles and the asynchronous decrease in endogenous progesterone concentrations. Hence, the efficacy of progestagen synchroni-
sation on the negative feedback regulation of LH secretion and the resultant effect on follicle growth towards the end of the treatment remains to be established.
The aim of the present experiment was to synchronise oestrus using different progestagen treatments in ewe lambs in which luteolysis was induced early in the
synchronisation period and to examine the effect on LH concentrations and the growth of the ovulatory follicle. We tested the hypothesis that in the absence of luteal
progesterone, ewes synchronised for 14 days with a single long-duration progestagen sponge will ovulate an older follicle and have higher LH pulse frequency than ewes
Ž synchronised for 14 days with three short-duration sponges removed and replaced every
. 5 days .
2. Materials and methods
2.1. Animals and treatments Ž
. During November, 10 Suffolk and 11 Texel cross ewe lambs 40.0 0.3 kg were
Ž .
maintained at pasture 53818’N where they had ad libitum access to water. A raddled, vasectomised ram was kept with the ewes to detect oestrus. The ewes were each fed 0.4
Ž .
kg of concentrates 19 crude protein per day. The ewes were presynchronised with an intravaginal sponge containing 60 mg
Ž .
medroxyprogesterone actate Veramix sponge, Upjohn . On Day 5 of the subsequent oestrous cycle ewes were assigned to one of three groups. Eight ewes received a new
sponge on Day 5 of the cycle that was left in place for 14 days and was removed on Day Ž
. 19 Long group . A second group of 8 ewes received a new sponge on Day 5 of the
cycle; the sponge was replaced with a new sponge on Days 10 and 15 and removed on Ž
. Day 19 Short group . Ewes in both the Long and the Short groups received prosta-
Ž
w
. glandin 0.5 ml i.m. Prosolvin ; Intervet, Cambridge, UK on Days 6 and 7 of the
Ž oestrous cycle. Finally, a group of five ewes did not receive any treatment Control
. Ž
. ovulation . All procedures including ultrasonography and blood sampling were li-
censed by the Department of Health and Children, Ireland, in accordance with the cruelty to animals act, 1876 and European Community Directive 86r609rEC.
2.2. Transrectal ultrasonography Follicular growth patterns were studied daily from Day 6 of the oestrous cycle by
Ž transrectal ultrasonography using a real time B mode scanner
Dynamic Imaging .
Concept MLV, Livingston, Scotland with a 7.5-MHz linear array transducer. The transducer was a standard I shape, typical of that used for transrectal examinations in
Ž .
cattle. The transducer was wrapped in a polythene tube 40 cm long, 15 mm o.d. so that it was rigid and thus could be inserted into the rectum and manipulated externally. The
ewes were restrained in a darkened room in a fostering crate on a raised platform. A lubricant gel was used as a coupling medium between the rectal wall and the transducer.
Images of the urinary bladder and the uterine horns were used in locating the position of the ovaries. The position and diameter of corpora lutea and follicles G 2 mm were
Ž .
recorded on a diagram each day Evans et al., 2000 . 2.3. Characteristics of follicular deÕelopment
An identified follicle was defined as a follicle that grew to at least 4 mm in diameter and was at least 3 mm in diameter for three or more days. Follicles with growth
characteristics not meeting these criteria were defined as unidentified follicles. The day of emergence was retrospectively traced as the day that follicles were first recorded with
a diameter of 2 or 3 mm. A follicle first identified at 4 mm was assumed to have been 3 mm in diameter the day before. The day that the largest follicle disappeared and was
later replaced by a corpus luteum was termed the day of ovulation. The interval between the day of emergence of the ovulatory follicle and the day of ovulation was referred to
as the life span of the ovulatory follicle.
The growth profile of identified follicles was tabulated and the number of waves of follicular growth produced by each animal was observed. Data from ewes with a similar
number of follicular growth waves that were in the same treatment group were amalgamated for presentation in graph form. This was done by plotting the aligned
growth profiles of the largest follicles in each wave to the mean day of emergence.
2.4. Blood collection and hormone analysis Ž
. Blood samples 7 ml were collected daily from Days 6 to 19 of the oestrous cycle by
Ž .
jugular venepuncture or until oestrus in control animals . The samples were stored at
Table 1 Ž
. Ž
Mean SEM oestrous cycle lengths and the characteristics of the ovulatory follicle in the control not .
Ž treated , short-duration a progestogen sponge was inserted on Day 5 of the oestrous cycle, replaced on Days
. Ž
10 and 15, and removed on Day 19; prostaglandin was given on Days 6 and 7 and long-duration sponge .
inserted on Day 5 and removed on Day 19 of the cycle; prostaglandin was given on Days 6 and 7 treatment groups
Ž .
Values in rows with no common superscript are different ab: P - 0.05; cd: P - 0.01 . Characteristic
Control Short-duration
Long-duration Ž
. Ž
. Ž
. ns 5
ns8 ns8
d c
c
Ž .
Cycle length days 15.80.3
22.00.3 21.50.3
Ovulatory follicle
d d
c
Ž .
Life-span days 6.80.6
5.10.5 12.10.6
d c
d
Ž .
Emergence day of cycle 10.00.7
17.50.7 10.40.8
b b
a
Ž .
Maximum diameter mm 6.30.4
6.30.2 7.40.2
a a
a
Ž .
Diameter at ovulation mm 6.30.4
6.30.2 6.70.2
Ž .
Ž .
Fig. 1. Mean SEM diameter of the corpus luteum, ovulatory follicle and largest subordinate follicle of
Ž .
Ž .
the ovulatory wave, oestradiol concentration and numbers of medium 4 and 5 mm and large G6 mm Ž
. Ž .
Ž follicles in control a; triangles; no treatment; ns 5 and sponge treated ewes b . Short-duration ewes circles;
. ns8 received a progestagen sponge on Day 5 of the oestrous cycle that was replaced on Days 10 and 15, and
Ž .
removed on Day 19 of the cycle. Long-duration ewes squares; ns8 had a sponge inserted on Day 5 and Ž
. removed on Day 19 of the oestrous cycle. Both short- and long-duration ewes received prostaglandin arrows
Ž .
on Days 6 and 7 of the cycle. Differences P - 0.05 between oestradiol concentrations and numbers of
follicles in long- and short-duration treated ewes on the same day are shown by q.
48C for 24 h and were then centrifuged at 1500 = g for 20 min at a temperature of 48C. The serum was decanted and stored at y208C until assayed for oestradiol concentra-
tions. On Day 18 of the cycle five ewes were selected at random from each of the Long-
Ž .
and Short-duration groups and blood samples 5 ml were taken every 12 min for 8 h. These blood samples were stored and processed in a similar manner to the daily blood
samples. The pulsatile pattern of LH in circulation was analysed using the PC Pulsar Ž
. pulse detection algorithm Gitzen and Ramirez, University of Illinois . The G parame-
Ž .
Ž .
ters G –G used were 4.4, 2.6, 1.92, 1.46 and 1.13 Merriam and Wachter, 1982 . The
1 5
pattern of LH secretion is described in terms of mean and basal concentration, pulse frequency and pulse amplitude.
2.5. Radioimmunoassays LH concentrations in frequently collected serum samples were assayed by validated
Ž .
y1
radioimmunoassay Cooke et al., 1997 . The sensitivity of the assay was 0.1 ng ml and the intra-assay and inter-assay coefficients variation for reference samples with
Ž .
Fig. 2. The mean SEM daily diameter of the largest follicle in each wave of follicular growth observed in Ž
. Ž
. Ž .
the control group ns 5, not treated . Two ewes ovulated a follicle from the second wave a , and three
Ž . ewes ovulated a follicle from the third wave b .
average concentrations of 0.41 ng ml
y1
, 1.68 ng ml
y1
and 3.52 ng ml
y1
included in each assay were 1.9 and 10.9, 2.7 and 7.4, 5.4 and 5.9, respectively.
Oestradiol concentrations in serum samples collected each day were assayed by Ž
. validated radioimmunoassay Prendiville et al., 1995 . The sensitivity of the assay was
0.2 pg per ml of serum. The intra-assay and inter-assay coefficients of variation for reference samples with average concentrations of 1.78, 2.76 and 5.37 pg ml
y1
included in each assay were 5.8 and 7.9, 7.6 and 7.0, 6.6 and 9.9, respectively.
Ž .
Fig. 3. The mean SEM daily diameter of the largest follicle in each wave of follicular growth observed in Ž
the short-duration treatment group ns8, 60 mg medroxyprogesterone acetate sponge inserted on Day 5 of .
Ž .
the oestrous cycle, replaced on Days 10 and 15, and removed on Day 19, boxes . Prostaglandin arrows was Ž
. Ž .
given on Days 6 and 7. One ewe ovulated a follicle from the third wave a , five ewes ovulated a follicle
Ž . Ž .
from the fourth wave b and two ewes ovulated a follicle from the fifth wave c of follicle growth.
2.6. Statistics Data were analysed using ANOVA or the general linear models procedure and
Ž .
included day, treatment, ewe and interactions where necessary SAS, Cary, NC .
Ž .
Multiple comparisons were made by the method of Tukey P - 0.05 . Prior to analyses, data were subject to Hartley’s test for heterogeniety of variance and logarithmic
transformations were done where necessary to yield homogeniety of variance. All values given are the mean SEM.
3. Results
