1. Introduction
Ž .
Release of oxytocin OT can be elicited at oestrus by mating and presence of the Ž
. male McNeilly and Ducker, 1972; Gilbert et al., 1991 and these high concentrations of
Ž .
OT may serve to facilitate gamete transport Wathes, 1984; Gilbert et al., 1992 . The pattern of OT release at mating appears to be highly species-dependent. In some species
the physical act of coitus is thought to be a relatively minor component in stimulating Ž
. the release of OT McNeilly and Ducker, 1972 and OT peaks may be provoked by the
Ž sensory and psychic stimuli occurring at the time of mating Campbell and Petersen,
. 1953; McNeilly and Folley, 1970; McNeilly and Ducker, 1972 . In the mare, mating is a
complex combination of sensory, psychic and mechanical stimuli involving teasing, mounting, intromission and ejaculation by the stallion. It is known that teasing stimu-
Ž .
lates OT peaks in the mare at mating Alexander et al., 1995 , but there is only limited Ž
. information on the pattern of OT release Walmsley, 1963; Alexander et al., 1995 .
Ž .
Prostaglandin F PGF
release is high in late dioestrus in mares, in association
2 a 2 a
Ž .
with luteolysis Goff et al., 1984 . Release of PGF can also be stimulated at other
2 a
Ž .
stages of the cycle by OT Goff et al., 1987 and there is a positive correlation between Ž
. plasma OT concentrations and PGF
release in nonpregnant mares Sharp et al., 1997 .
2 a
Therefore, any increase in OT at mating in mares might be accompanied by release of Ž
PGF . Both PGF and OT have been implicated as ecbolic agents in mares Troedsson
2 a 2 a
. et al., 1995 . Persistent mating-induced endometritis is a major cause of subfertility in
mares. Susceptibility to this condition is associated with low myometrial contractility Ž
. Troedsson et al., 1993 . Increases in concentrations of PGF
and OT around the time
2 a
of mating may therefore be important not only in gamete transport, but in stimulating uterine contractile activity after mating, which serves to evacuate collections of inflam-
Ž matory uterine fluid and cellular debris in genitally normal mares Hughes and Loy,
. 1975 .
We hypothesised that mares susceptible to endometritis, with delayed uterine clear- ance after mating may have defective release of ecbolic hormones. However, there is no
detailed information on endocrine events occurring around the time of mating and other intrauterine interventions in genitally normal mares. Therefore, in the present study, we
measured the release patterns of the ecbolic hormones OT and PGF at the time of
2 a
mating in the mare and some hours following mating when clearance of uterine contamination may occur. We also investigated the effect of a range of stimuli
associated with mating on OT and PGF release.
2 a
2. Materials and methods
2.1. Animals Eleven fertile mares, aged 5 to 15 years and weighing between 350 and 480 kg, were
used. These mares had been classified as genitally normal, according to their reproduc- tive history, negative endometrial cytology and culture, and endometrial biopsy scores of
Ž .
1–2A Kenney and Doig, 1986 . Oestrus was detected by teasing with a stallion,
combined with transrectal ultrasonographic examination of the genital tract. When the mare responded positively to teasing and uterine oedema was present, with a follicle of
at least 35 mm present on the ovaries, the mare was considered to be in oestrus. Ovulation was detected ultrasonographically by the disappearance of the follicle and the
presence of a corpus haemorrhagicum on the ovary. Day of ovulation was designated as day 0. A stallion with good libido was used in experiments 1 and 2.
2.2. Experimental procedures The same blood sampling protocol was used in all experiments. On the day of the
Ž .
experiment, an indwelling cannula 13 gauge, Presidio Medico, Ecouen, France was placed in the jugular vein aseptically under local anaesthesia. Blood samples were
collected at 2-min intervals for 30 min before, during, and 1 h after the application of the stimulus, and then for another hour, at 5-min intervals.
Ž .
Five different stimuli were applied. In experiment 1, oestrous n s 5 and dioestrous Ž
. Ž
. day 7; n s 5 mares were teased by the stallion for 6 min three samples in teasing
stocks. The stallion was allowed to nuzzle and nip the mare. In experiment 2, oestrous mares were teased as in experiment 1, but in this case teasing was followed immediately
Ž .
Ž .
by mating n s 5 . In experiment 3, the genital tracts of oestrous mares n s 4 were manually manipulated to simulate the individual stimuli associated with mating. All
stimuli applied were exaggerated in duration in order to intensify the response to the particular stimulus. The mares’ external genitalia were actively massaged for 6 min
Ž
. three samples , while washing three times with tamed povidone iodine solution, after
which the operator inserted a sterile gloved lubricated hand into the vagina, moving it Ž
. carefully palindromically for 4 min two samples , distending the vaginal walls without
coming into contact with the cervix. The cervix was then massaged and manipulated for Ž
. 2 min one sample . In experiment 4, the mechanical effect that the introduction of fluid
into the uterus andror uterine distension has on hormonal release in oestrous mares Ž
. n s 5 was determined by intrauterine infusion of 500 ml phosphate buffered saline
Ž .
PBS; pH 7.0 . This high volume was chosen from a preliminary study in which cross-dimensional areas had been calculated from transrectal ultrasound scans of differ-
Ž .
ent volumes of infused buffer Nikolakopoulos and Watson, 1999 . It was seen that at this volume the fluid did not merely remain in one pocket, but distended and contacted a
Ž .
significant part of the endometrium Nikolakopoulos, 1999 . Additional blood samples were collected every 15 min between 16 and 18 h after mating for measurement of
PGFM concentrations to determine whether the high uterine myoelectrical activity Ž
reported at this time after bacterial infusion in genitally normal mares Troedsson et al., .
1993 is due to the release of prostaglandins. 2.3. Sample handling
Blood samples were collected into evacuated heparinised tubes and placed on ice until separation. The samples were centrifuged at 2000 = g for 15 min at 48C. An
Ž .
aliquot of plasma was acidified with 10 M acetic acid 10 mlrml plasma for oxytocin assay. All samples were frozen at y708C and subsequently stored at y208C until
assayed.
2.4. Oxytocin assay Ž
Oxytocin was extracted from 2 ml plasma using C SepPak cartridges Waters
18
. Chromatography, Milford, MA, USA and the radioimmunoassay carried out in dupli-
Ž .
cate samples as described by Thornton et al. 1986 using a previously characterised Ž
. antiserum Sheldrick and Flint, 1981 . The extraction recovery rate was 74.8. The
detection limit for the assay was 0.8 pgrml. The intra- and inter-assay coefficients of variation were 7.5 and 11.1, respectively.
2.5. 15-Keto-13,14-dihydro-PGF assay
2 a
The release of PGF was monitored by measuring its main initial plasma metabolite
2 a
Ž .
Ž 15-keto-13,14-dihydro-PGF
PGFM . Duplicate samples of unextracted plasma 0.2
2 a
. ml were assayed. Before the addition of antibody and radioactive tracer, 0.3 ml 0.25
bovine gamma globulin in buffer was added and the tubes were heat-treated for 30 min Ž
. at 458C. The antibody cross-reacted with 15-keto-PGF
16.0 , 13,15-dihydro-PGF
2 a 2 a
Ž .
Ž .
4.0 , and 15-ketodihydro-PGE 1.7 . Other prostaglandins tested cross-reacted less
2
than 0.1. The detection limit of the assay was 20 pgrml. The intra- and inter-assay coefficients of variation were 8.5 and 14, respectively.
2.6. Statistical analysis Mean baseline hormone concentrations were calculated from the average of the
Ž .
values obtained prior to the application of the stimulus Time 0 and mean hormone concentrations were calculated for every 30-min interval thereafter. Concentrations
below the detection limit of the assays were designated as equivalent to the detection limit of the respective assay. In the case of teasing and manipulation of the genital tract,
mean stimuli values for both hormones were obtained from the samples corresponding to the time of the different stimuli application. During natural service and uterine
infusion, mean OT values were obtained from the samples corresponding to the mean OT concentrations of the peak immediately after the application of the stimulus until OT
concentrations returned to baseline levels. Mean baseline OT and PGFM concentrations from all experiments were compared using one-way ANOVA test. Mean baseline values
and mean stimuli values for both OT and PGFM, for each experiment, were compared using a two sample t-test. In experiment 3, mean stimuli values for both OT and PGFM
were compared with mean baseline values separately for each stimulus. The difference in magnitude of OT responses between different stimuli was compared using one-way
ANOVA tests.
Because of the pulsatile nature of OT release and its short half-life, responses were assessed for individual mares. A mare was considered to have a positive OT response
when the mean concentration of the peak immediately after the application of the stimulus exceeded the mean baseline concentrationsq 2 = SD. Mean PGFM concentra-
Ž .
tions were calculated for each mare as above 30-min intervals and a positive response
was recorded when the increase for each mare, at any 30-min interval after the stimulus application, exceeded the mean baseline concentrationsq 2 = intra-assay coefficient of
variation.
3. Results
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