G.E. Mann, G.E. Lamming Animal Reproduction Science 64 2000 171–180 173
effects of the level of preovulatory oestradiol on subsequent oxytocin receptor concentra- tions and activity using a hormone treated ovariectomised cow model.
2. Materials and methods
2.1. Experimental animals Two studies were undertaken in mature, ovariectomised Blue-Grey cows. All cows had
been ovariectomised between 6 and 7 years previously. Since then cows had been used in between four and five progesteroneoestradiol replacement experiments. Prior to the current
study, cows had not been used for at least 6 months, and were then randomly allocated to treatment groups. In the current study, all cows were administered a 2 day 17b-oestradiol
treatment regimen to replace preovulatory oestradiol secretion during a simulated follicular phase. All treatments are described relative to the second day of oestradiol treatment, defined
as day 0, on which all cows exhibited behavioural oestrus mounting activity; standing to be mounted; vaginal “sliming”.
2.2. Experimental design 2.2.1. Study 1
In this study, three groups of four cows were treated with different levels of 17b-oestradiol Sigma, Fancy Road, Poole, UK, administered by i.m. injection in corn oil at 8 h intervals
over a 48 h period according to Table 1 to simulate three levels of preovulatory oestradiol production. The effect of the level of oestradiol on subsequent oxytocin receptor decrease
was assessed by measuring oxytocin binding activity in endometrial biopsy samples col- lected on days 2, 4 and 6 following oestrus day 0. Throughout the study, plasma samples
were collected to monitor plasma oestradiol concentrations.
2.2.2. Study 2 In this study, the same three levels of oestradiol were administered to produce three levels
of simulated preovulatory oestradiol secretion. However, in this study cows were then treated with progesterone by i.m. injection in corn oil twice daily, from days 3 to 6 following oestrus
Table 1 Quantities of oestradiol mg per i.m. injection administered to ovariectomised cows during simulated follicular
phases Time h
High n = 4 Medium n = 4
Low n = 4 50
25 12.5
8 100
50 25
16 150
75 37.5
24 200
100 50
32 200
100 50
40 200
100 50
174 G.E. Mann, G.E. Lamming Animal Reproduction Science 64 2000 171–180
day 3: 15 mg × 1; day 4: 30 mg × 2; day 5: 60 mg × 2; day 6: 90 mg × 1 to simulate a post-ovulatory progesterone rise. The potential for a premature luteolytic signal was then
determined on day 6 by measuring plasma concentrations of 13,14-dihydro-15-keto-PGF
2
a
PGFM, the principle metabolite of PGF
2
a
, in samples collected before and after a 50 i.u. oxytocin challenge.
2.3. Blood sampling, biopsy collection and oxytocin challenge Prior to the start of oestradiol treatment, a jugular vein of each animal was cannu-
lated under local anaesthesia 2 ml lignocaine s.c. as Lignovet 2, C-Vet, Bury St Ed- munds, UK with a 30 cm indwelling jugular catheter Secalon universal tubing, BOC
Health Care, Swindon, UK using a 12 gauge needle and guide wire. Cannulae were then maintained for the duration of the experiment, and used for the collection of all blood
samples.
Endometrial biopsies were collected via a trans-cervical technique Mann and Lamming, 1994. During biopsy collection, animals were first sedated by an i.m. injection of 20 mg
xylazine Rompun; Bayer U.K. Ltd, Science Park, Milton Road, Cambridge and the biopsy forceps were guided through the cervix by trans-rectal manipulation. Once in the uterus,
a single sample of 300–600 mg endometrial tissue was collected. Animals were then ad- ministered prophylactic antibiotic Duplocillin LA; Mycofarm UK, Science Park, Milton
Road, Cambridge. Immediately following collection, samples were snap frozen in liquid nitrogen and then stored in liquid nitrogen until processed for determination of endometrial
oxytocin binding.
To monitor endometrial responsiveness to oxytocin, cows were injected with a single iv. bolus of 50 i.u. oxytocin Hoechst UK Ltd, Walton Manor, Milton Keynes in 5 ml saline,
flushed in with a further 5 ml saline. Plasma concentrations of PGFM were measured in blood samples collected at 20 min intervals for 1 h before the injection of oxytocin, and then
at 10 min intervals for 1 h after the challenge. All samples were collected into heparinised tubes, centrifuged at 1500 × g for 10 min and the plasma stored at −20
◦
C. 2.4. Radioimmunoassays
Plasma samples were assayed for PGFM after extraction with acidified diethyl ether by radioimmunoassay Kaker et al., 1984. Intra- and inter-assay coefficients of variation were
12.6 and 14.3, respectively. Progesterone was measured in plasma samples after extraction with petroleum ether by radioimmunoassay Haresign et al., 1975. Intra- and inter-assay
coefficients of variation were 8.8 and 12.4, respectively. Oestradiol was measured in plasma using a modified radioimmunoassay kit Serono Diagnostics Ltd, Woking, Surrey,
UK Mann et al., 1995. Intra- and inter-assay coefficients of variation were 8.5 and 11.1, respectively. To determine oxytocin binding, sub-cellular membrane fractions were prepared
according to the method of Sheldrick and Flint 1985. Oxytocin binding by the membrane preparations was then determined using the binding assay of Sheldrick and Flint 1985,
modified for use in bovine samples Jenner et al., 1989. Results were expressed as fmol oxytocin bound per mg total protein Lowry et al., 1951. The sensitivity of the assay was
20 fmol oxytocin bound per mg total protein.
G.E. Mann, G.E. Lamming Animal Reproduction Science 64 2000 171–180 175
2.5. Statistical analysis Plasma concentrations of oestradiol were analysed by repeated sample analysis of vari-
ance with group and day as main factors. Changes in plasma concentration of PGFM follow- ing oxytocin challenge were analysed by repeated sample analysis of variance with group
and time as main factors. Endometrial oxytocin binding results were analysed by analysis of variance with group and day as the main factors. The effects of level of oestradiol on
oxytocin binding were analysed by regression analysis with mean oestradiol concentration on day 0 and oxytocin receptor concentration on day 6 as factors. The effects of level of
oestradiol on oxytocin challenge response on day 6 were analysed by regression analysis with mean oestradiol concentration on day 0 and mean PGFM increase on day 6 expressed
as percentage increase above pre-treatment baseline as factors.
3. Results
