www.elsevier.comrlocateranireprosci
Release of oxytocin and prostaglandin F
2aaround
teasing, natural service and associated
events in the mare
E. Nikolakopoulos
a, H. Kindahl
b, C.L. Gilbert
c, J. Goode
c,
E.D. Watson
a,)a
Department of Veterinary Clinical Studies, UniÕersity of Edinburgh, Easter Bush Veterinary Centre, Roslin,
Midlothian, EH25 9RG, Scotland, UK
b
Department of Obstetrics and Gynaecology, Swedish UniÕersity of Agricultural Sciences, Uppsala, Sweden
c
The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
Received 1 December 1998; received in revised form 8 February 2000; accepted 20 March 2000
Abstract
Ž .
Mating has been shown in many species to provoke the release of oxytocin OT . In our study,
Ž .
various stimuli were applied to mares to study release of OT and prostaglandin F2a PGF2a associated with mating. Blood samples were collected from mares around the time of teasing both in oestrus and dioestrus and at mating. For comparison, blood samples were also collected at the time of manual manipulation of the genital tract and after intrauterine infusion of 500 ml
Ž .
phosphate buffered saline PBS . Additional samples were collected 16 to 18 h after mating. Mating caused a significant increase in OT in all mares and teasing caused a significant OT response in 6 of 10 oestrous and 3 of 5 dioestrous mares. However, mating and teasing had no
Ž .
significant effect on concentrations of 15-keto-13,14-dihydro-PGF2a PGFM . Manual manipula-tion of the clitoris, vagina and cervix caused significant OT release in all mares and intrauterine infusion of 500 ml PBS caused significant OT release in three of the five mares. However, only one mare had a significant PGF2a response during manual manipulation and only one responded positively to intrauterine infusion of 500 ml PBS. We concluded that events around mating, including stimulation of the genital tract and uterine distension, often caused an increase in circulating concentrations of OT but only rarely in PGFM. q2000 Elsevier Science B.V. All rights reserved.
Keywords: Horse; Oxytocin; Prostaglandin; Mating; Teasing
)Corresponding author. Tel.:q44-131-650-6235; fax:q44-131-650-6588.
Ž .
E-mail address: [email protected] E.D. Watson .
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
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 F2a PGF2a release is high in late dioestrus in mares, in association
Ž .
with luteolysis Goff et al., 1984 . Release of PGF2a can also be stimulated at other
Ž .
stages of the cycle by OT Goff et al., 1987 and there is a positive correlation between
Ž .
plasma OT concentrations and PGF2a release in nonpregnant mares Sharp et al., 1997 . Therefore, any increase in OT at mating in mares might be accompanied by release of
Ž
PGF . Both PGF2a 2a and OT have been implicated as ecbolic agents in mares Troedsson
.
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
ŽTroedsson et al., 1993 . Increases in concentrations of PGF. 2a and OT around the time 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 PGF2a at the time of 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 PGF2a release.
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, negareproduc-tive endometrial cytology and culture, and endometrial biopsy scores of
Ž .
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 ns5 and dioestrous
Žday 7; ns5 mares were teased by the stallion for 6 min three samples in teasing. Ž .
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 ns5 . In experiment 3, the genital tracts of oestrous mares ns4 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
Žthree 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
Žns5 was determined by intrauterine infusion of 500 ml phosphate buffered saline. ŽPBS; 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 .
2.4. Oxytocin assay
Ž
Oxytocin was extracted from 2 ml plasma using C18 SepPak cartridges Waters
.
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-PGF2a assay
The release of PGF2awas monitored by measuring its main initial plasma metabolite
Ž . Ž
15-keto-13,14-dihydro-PGF2a PGFM . Duplicate samples of unextracted plasma 0.2
.
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-PGF2a 16.0% , 13,15-dihydro-PGF2a
Ž4.0% , and 15-ketodihydro-PGE. 2Ž1.7% . Other prostaglandins tested cross-reacted less.
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 concentrationsq2=SD. Mean PGFM
concentra-Ž .
was recorded when the increase for each mare, at any 30-min interval after the stimulus application, exceeded the mean baseline concentrationsq2=intra-assay coefficient of variation.
3. Results
3.1. Experiment 1
For the evaluation of responses to teasing, teasing periods before mating were also included. All oestrous teasings were accompanied by a display of oestrous behaviour such as posturing and clitoral winking; while during all dioestrous teasings, the mares demonstrated aggressive behaviour and rejected the stallion. Teasing caused a significant
ŽP-0.05 increase in OT concentrations in both oestrus and dioestrus Fig. 1 . There. Ž .
Ž .
was no significant difference P)0.1 either between the number of mares responding
Ž . Ž .
to teasing with increased OT release in oestrus 6r10 and dioestrus 3r5 or in the magnitude of response. In both dioestrous and oestrous teasing periods, except those followed by mating, mean OT concentrations declined to baseline values within 30 min
ŽFig. 1 . On three occasions, an OT peak was observed immediately prior to the.
initiation of teasing, coinciding with the entrance of the stallion into the mare’s visual field. Although teasing had no significant effect on mean PGFM concentrations in
Ž .
oestrus Fig. 1 , elevations were observed in two of the five sampling periods after teasing. None of the dioestrous mares released significant amounts of PGF2a at teasing
ŽFig. 1 ..
Ž . Ž . Ž .
Fig. 1. Oxytocin and PGFM at teasing in 10 oestrous mares and in 5
Ž .
dioestrous mares. Note that only 5 oestrous mares were sampled after teasing. Asterisks represent a
Ž .
3.2. Experiment 2
All five mares had a positive OT response during natural service and mean stimuli
Ž . Ž .
values were significantly higher P-0.05 than baseline values Fig. 2 . Furthermore, in two mares in which false mounting occurred without intromission prior to successful mating, an oxytocin peak was observed. Mean OT concentrations after mating declined to baseline values within 30 min after the application of the stimulus. Mating had no
Ž .
significant effect on mean PGFM concentrations Fig. 2 , but minor elevations were observed in three of the five mares after mating. There was no detectable PGFM in the
Ž .
samples collected 16–18 h after natural service data not shown .
3.3. Experiment 3
Manipulation of the genital tract consisted of three distinct stimuli to which the OT and PGFM response varied. Active massage of the clitoris and external genitalia
Ž .
significantly P-0.05 increased OT concentrations in two of the four mares. Both
Ž .
distension of the vaginal walls and cervical stimulation significantly increased P-0.05 mean OT concentrations. In only one of the mares did PGFM levels reach statistical significance when compared to mean baseline concentrations in response to manipula-tion of the genital tract. In all mares, mean OT concentramanipula-tions returned to baseline levels
Ž .
within 1 h of the application of the stimuli Fig. 3 . Mean PGFM concentrations in the one responding mare did not return to baseline levels by the end of blood sampling, 2 h after the application of the stimuli.
Ž . Ž .
Fig. 2. Oxytocin clear bars and PGFM shaded bars in 5 oestrous mares around mating. Asterisk represents a
Ž .
Ž . Ž .
Fig. 3. Oxytocin clear bars and PGFM shaded bars levels around manipulation of the genital tract in 4
Ž . Ž
oestrous mares. Active massage of the vulva and clitoris stimulus 1 , distension of the vaginal walls stimulus
. Ž .
2 and cervical manipulation stimulus 3 are noted. Asterisk represents a significant increase from baseline
Ž .
concentrations y30–0 min .
3.4. Experiment 4
The intrauterine infusion of 500 ml PBS provoked a significant OT response
ŽP-0.05. ŽFig. 4 . Mean OT concentrations returned to baseline levels within 30 min.
from the time of infusion. There was a positive PGFM response in only one mare.
Ž . Ž .
Fig. 4. Oxytocin clear bars and PGFM shaded bars in 5 oestrous mares after intrauterine infusion of 500 ml
Ž .
4. Discussion
In the present study, we have shown that numerous stimuli associated with mating can result in oxytocin release in genitally normal mares. The effect of central OT on reproductive behaviour has been reported to be species-specific and dependent on
Ž
circulating concentrations of sex steroids Caldwell et al., 1989; Carter, 1992; Insel et
.
al., 1997 . It has been shown in other species that external stimuli, such as olfactory
ŽMcNeilly and Folley, 1970; McNeilly and Ducker, 1972 , visual McNeilly and Folley,. Ž
. Ž .
1970; McNeilly and Ducker, 1972 , and tactile Schams et al., 1982 signals associated with teasing are directly responsible for the release of OT. In the mare, posturing is an indicator of oestrus and is an indispensable part of mating behaviour. It is provoked by visual, tactile and acoustical stimulation during teasing. In the present study, although oestrous teasing was associated with sexually receptive behaviour in all mares, OT peaks were detected in only some of the mares. In agreement with the present study, Madill et
Ž .
al. 1988 reported that not all oestrous mares released OT in response to teasing. It is possible however that peaks of OT were missed in the present study by sampling peripheral blood, as concentrations of OT are eight fold higher in blood collected from
Ž .
the intracavernous sinus of mares Vanderwall et al., 1998 . Exhibition of oestrus in the mare seems to be primarily dependent on the circulating concentrations of steroid hormones since, in our study, teasing at day 7 of dioestrus, when progesterone levels are high, provoked an OT response that was accompanied by rejection of the male. These observations support the concept that the steroid environment can modify responsiveness
Ž .
to somatosensory stimuli mediated by OT in the brain Caldwell et al., 1996 .
The physical stimulus of coitus per se is a relatively minor component in stimulating
Ž
the release of OT in the oestrous goat McNeilly and Folley, 1970; McNeilly and
. Ž .
Ducker, 1972 and cow Schams et al., 1982 . By contrast, coitus-evoked OT release
Ž . Ž .
was reported in sows Claus et al., 1989 and in women Carmichael et al., 1987 . It has been suggested that these inter-species differences are related to the degrees of physical
Ž .
stimulation caused by the male Schams et al., 1982; Claus et al., 1989 . The mating sequence in the mare includes a total mount time of 20 to 30 s with an insertion time before ejaculation of 10 to 15 s during which the stallion thrusts from six to nine times
ŽMcDonnell, 1992 . In the mare, the oestrous cervix is relaxed, permitting dilation by.
the greatly distended glans penis, thus allowing intrauterine deposition of semen. Therefore, it might be expected that OT would be elevated in the mare at mating.
Ž .
Walmsley 1963 detected a rise in plasma OT after mating in only one of the four
Ž .
mares studied and Alexander et al. 1995 reported an elevation of OT levels in two of the three mares in single samples collected within 5 min after mating. In our study, all five mares showed substantial OT release around the time of mating in agreement with
Ž .
the results of Madill et al. 1998 . In two mares, which had to be mounted twice by the stallion, the false mount was associated with release of OT on both occasions. Similarly,
Ž . Ž .
in the cow VanDemark and Hays, 1952 and the ewe Lightfoot, 1970 , mounting without intromission caused an immediate increase in the frequency and amplitude of uterine contractions possibly reflecting OT release.
genitalia and clitoris provoked OT release in two mares. Vaginal distension and cervical
Ž
manipulation have been reported to release OT in other species Roberts and Share,
.
1969; Blank and De Bias, 1977; Schams et al., 1982; Kendrick et al., 1991 as well as in
Ž
the mare Betteridge et al., 1985; Aurich et al., 1996; Sharp et al., 1997; Vanderwall et
.
al., 1998 . In our study, vaginal distension, cervical stimulation and intrauterine infusion of 500 ml PBS provoked a significant OT response. On several occasions shortly after PBS infusion, the mares adopted a urinating posture or were actually expelling fluid,
Ž .
often coinciding with OT peaks data not shown . This observation is consistent with the findings of another study, where reflux of infused fluid often occurred within 10 to 15
Ž .
min post-infusion Jones, 1995 and it appears in our study that uterine distension provokes uterine contractions, probably elicited by OT release. In our mares, vaginal distension provoked the greatest and most consistent release of OT compared with the
Ž .
other stimuli P-0.05 . Although manipulation of the genital tract caused a signifi-cantly greater OT response than mating this is probably due to the longer period of time
Ž12 min that the stimuli were applied in comparison to the duration of mating in the.
Ž .
mare 2 min; McDonnell, 1992 . The results of our study demonstrated a gradation in the OT response depending on the duration and severity of the applied stimulus.
Peaks of OT were highest at the time of the stimulus application, and declined rapidly reaching baseline levels by 30–60 min in all experiments. Interestingly, where there was a measurable PGFM response, elevations tended to follow the OT peaks, indicating a possible temporal correlation between the two hormones as already suggested in mares
ŽVanderwall et al., 1998 . No PGFM response was detected at two of the five mating.
and three of the five teasing episodes. Also, there was no prostaglandin release 16 to 18 h after mating, although increased myoelectrical activity has been reported at that period
Ž
of time after intrauterine infusion of bacteria to genitally normal mares Troedsson et al.,
.
1993 . However, increased uterine activity could be due to local prostaglandins, released
Ž .
from the inflamed endometrium Watson, 1989 and detected in uterine flushings
ŽWatson, 1987 , which may not reach the circulation in detectable concentrations..
Ž .
Betteridge et al. 1985 showed in dioestrous mares that vaginal distension, uterine manipulation and cervical dilation increased circulating PGFM levels in 50% of the mares and that rectal palpation and washing of the vulva did not affect PGFM concentrations. In our study, only one oestrous mare had significant PGFM response after manipulation of the genital tract. It has previously been observed that individual mares appear to have different capacities to release PGF2a in response to stimuli applied
Ž . Ž .
to the genital tract Kask et al., 1997 . Furthermore, Goff et al. 1987 reported that during oestrus there was a dramatic decline in the PGFM response to exogenous OT possibly as a result of the low endometrial oxytocin receptor levels at this stage of the
Ž .
cycle Sharp et al., 1997 . It also appears that the endometrium may require a period of exposure to progesterone before it has the capacity to release large quantities of PGF2a
ŽVernon et al., 1981 . This may account for failure of PGF. 2a release in a number of oestrous mares and the low level of release in responding mares in comparison with
Ž .
concentrations measured at luteolysis Goff et al., 1987 .
minority of mares over all experiments. Future studies should investigate patterns of release of ecbolic hormones after similar stimuli applied to mares with delayed uterine clearance.
Acknowledgements
We thank Ms. S. Thomson for help with sample processing, Ms. M. Jordan for taking excellent care of the research mares and Ms. L. Jensen for assaying PGFM samples. We would also like to thank Dr. B. Worton and Mr. O. Papasouliotis from STATLAB, the University of Edinburgh for statistical support, the University of Edinburgh Veterinary Faculty Research Fund and the Swedish Council for Forestry and Agricultural Research for partial financial support of the study.
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