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Uterine activity, sperm transport, and the role of boar stimuli around insemination in sows
Theriogenology 63 (2005) 500–513 www.journals.elsevierhealth.com/periodicals/the
Uterine activity, sperm transport, and the role of boar stimuli around insemination in sows P. Langendijk*, N.M. Soede, B. Kemp Department of Animal Science, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands
Abstract This paper describes changes in spontaneous myometrial activity around estrus, factors that affect myometrial activity, and the possible role of uterine contractions in the process of (artificial) insemination, sperm transport and fertilization. Myometrial activity in the sow increases during estrus. The activity is myogenic in origin, but several factors have been shown to affect myometrial activity. Natural mating stimulates uterine contractions through several mechanisms. The presence of a boar, rather than the act of mating, induces central oxytocin release in the sow and thus increases uterine activity. Estrogens in the ejaculate of a boar can trigger prostaglandin release by the endometrium and thus increase uterine activity. Tactile stimulation of the genital tract (cervix) or tactile stimulation of the back and flanks of the sow during artificial insemination does not cause a release of oxytocin. There is hardly any evidence for the effects of these latter stimuli on uterine activity, and if they are present at all, the effects are very small. Evidence for the effects of synthetic boar odor on oxytocin release and/or uterine activity is inconsistent. The mere presence of a boar during insemination, in contrast, clearly stimulates uterine activity through the release of oxytocin. Hormonal stimulation (intrauterine) of uterine activity with estrogens, prostaglandins, or oxytocins before, during or after insemination generally improves fertilization rate, especially in situations with reduced fertility. Therefore, uterine contractions are believed to play an important role in the transport of sperm cells to the oviducts after insemination. Whether uterine contractions are absolutely necessary for sperm transport through the uterine horns, however, is not clear. Intensive stimulation of uterine contractions using hormones can also reduce the fertilization rate, probably by increasing the reflux of sperm cells during insemination. In this respect, the presence of a boar during
* Corresponding author. Tel.: +31 317 483197; fax: +31 317 485006. E-mail address: [email protected] (P. Langendijk). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.09.027
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AI seems more adequate, as only sows with a low level of uterine activity show an increase in uterine activity in response to this stimulus. # 2004 Elsevier Inc. All rights reserved. Keywords: Uterine contractions; Sperm transport; Boar stimuli; Fertilization; Insemination
1. Introduction During mating and artificial insemination in sows, semen is deposited intra-cervically. However, due to the volume, the bulk of the semen is flushed directly into the lumen of the uterine body, immediately before the cervix. Therefore, semen deposition is often referred to as intrauterine. From the site of deposition, sperm cells must be distributed over both horns and transported to the tubal end of the horns. There, a relatively small number populates the utero-tubal junction and the first part of the oviduct, which serve as a sperm reservoir. The transport of sperm cells through the uterine horns is believed to be a passive process, in which intrinsic sperm cell motility plays no part. This passive process probably depends on the flow of sperm cell-containing fluids in the uterine lumen, driven by gravitational force and uterine contractility. In the present paper, the available literature on uterine activity around estrus in sows is reviewed, and the role of uterine activity in the transport of sperm cells and the effects of boar stimuli are discussed.
2. Spontaneous uterine activity Uterine activity around estrus in sows has been studied using several invasive methods (Table 1), either by applying strain gauges to the uterine wall to record the stretch exerted by the uterine musculature [1,2] or by applying electrodes to record electromyographic activity [3–6]. A drawback of both strain gauges and electromyography is that these methods do not directly record pressure changes in the uterine lumen, since electrical activity does not necessarily induce muscular contraction. This translation depends on the sensitivity of the myometrium to electrical triggering, which in turn depends on the physiological status of the myometrium [2]. Several types of electrical activity, as defined by Scheerboom et al. [2], all coincide with myometrial contractions as recorded with strain gauges. However, it is not clear from the study by Scheerboom et al. [2] how electrical activity correlates quantitatively with myometrial contraction. Intraluminal pressure changes are a more direct indication of muscular contraction, and intraluminal pressure changes after all are the driving force for transport of fluids (e.g. semen) from one place to the other. Do¨ cke and Worch [7] recorded uterine intraluminal pressure using a fluid-filled balloon inserted into the cervical end of the uterus. There are no other reports on the use of non-invasive techniques to record intraluminal pressure in sows. Over the past few years, we have developed a non-invasive technique [8] by adapting a device developed for nonsurgical embryo transfer in pigs [9]. With this device it is possible to pass the cervix and introduce an open-ended, fluid-filled catheter into the caudal end of the uterine horns to
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Table 1 Myometrial activity during and around estrus, as reported by different authors, using different methods Author and method
Parameter a
Diestrus
Proestrus
Estrus
Scheerboom (1987) EMGb
Frequency of MC Amplitude
4/h 115 mV
14/h 227 mV
10/h 870 mV
Claus et al. (1989) EMG
Frequency of burstsc Duration of bursts Amplitude Luteal phase activity below detection level
90/h 2s 180 mV
17/h 22 s 1720 mV
20/h 32 s 2040 mV
Brussow et al. (1988) EMG
Frequency of APd Relative duratione
8/h 22%
29/h 25%
Bower (1974) Strain gauges
Frequency of contractions Force of stretch
0/h 0g
8/h 14 g
15/h 15 g
Zerobin and Spo¨ rri (1972)f Intraluminal pressure
Frequency of contractions Amplitude of contractions Duration of contractions
<8 mmHg
60/h <15 mmHg 25 s
21–30/h 35–45 mmHg Maximum 60 s
Langendijk et al. (2002a) Intraluminal pressure
Frequency of contractions Amplitude of contractions Duration of contractions
15/h 20 mm Hg 60 s
22/h 40 mm Hg 60 s
a b c d e f
MC = myoelectric Complex. EMG = electromyography. Bursts: bursts of electrical activity. AP = action potentials. Relative duration: duration of electrical activity, relative to measuring time. For Zerobin and Spo¨ rri, approximate figures are given because of a lack of quantitative information.
record intraluminal uterine pressure. With this technique, we have studied spontaneous myometrial activity around estrus. Two to four days before estrus, myometrial activity is low in terms of frequency and amplitude of contractions [8]. A percentage of sows (around 50%) does not show any contractility at all around this time, and the sows that do show contractility tend to have a low frequency and amplitude of contractions, compared to the situation during estrus (Table 1). Approaching estrus, the percentage of sows showing myometrial activity increases, and the frequency and amplitude of contractions increases too. During estrus, all sows show myometrial activity and the frequency and amplitude of contractions are maximal. After estrus, uterine activity decreases again. Using the non-invasive technique, data could be collected in a relatively large number of sows [8], which enabled us to study the variation between sows. The variation between sows during estrus is huge: the frequency of contractions ranges from 6 to 40/h and average amplitude per sow ranges from 16 to 57 mm Hg. Nevertheless, the difference between sows is consistent over the days around estrus, i.e., sows with a relatively high level of uterine activity during the days before estrus also show a relatively high level of uterine activity during estrus. This indicates a sow-dependent variation throughout the estrous cycle. Variation between sows over cycles also exists: for cyclic sows in which more than one estrous cycle was monitored, the variation between sows explained 60% of the total variation (cycle
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variation + sow variation) in contraction frequency during estrus (Langendijk et al., unpublished results). Variation in uterine activity during the estrous cycle has also been observed by other authors. The force of contraction increases during estrus, regardless of what method is used [1–3,5,6], but not all authors report an increase in the frequency of contractions during estrus (Table 1). However, quantitative comparisons between studies are difficult because the site of recording, the method of recording (electrical activity, muscle stretch and intraluminal pressure), and the definition of parameters differ between studies. With electromyographic observations, a lower frequency of electrical activity is sometimes reported during estrus than outside of estrus [2,6]. However, the lower frequency of electrical activity during estrus does not necessarily correlate with lower contractile activity (see data of Scheerboom et al. [2] on electromyography and strain gauges). During estrus, the amplitude and duration of electrical bursts are more pronounced [6] and the myometrium is more sensitive to electrical input. Thus, a lower frequency of electrical bursts (that are more pronounced) during estrus can still result in a higher frequency of contractions. Nevertheless, Zerobin and Spo¨ rri [3] also observed a decrease in the frequency of contractions during estrus, and they used intraluminal catheters. However, the contractions during estrus (low frequency, high amplitude) were of much higher amplitude (35–45 mm Hg) compared to the contractions outside estrus (high frequency, low amplitude; up to 3 mm Hg), and with different criteria (a higher threshold to accept a pressure fluctuation as a contraction) a lower frequency of contractions would probably have been recorded outside estrus. In conclusion, the amplitude of contractions is increased during estrus and the frequency of contractions is highly dependent on the criteria used to define contractility. However, ‘functional’ uterine activity, in terms of sperm transport, is probably increased during estrus. The variation in myometrial activity throughout the estrous cycle and between sows probably depends mainly on the tissue and plasma levels of estrogen and progesterone. Progesterone inhibits and estrogen increases uterine activity. From studies on other species than the pig, it appears that estrogen increases gap junctional coupling between myometrial cells [10], the myosin content of myometrial cells [11], and the pacemaker activity of myometrial cells [12]. Data from Hoang-Vu [13] indicate that estrogen levels, but also the estrogen/progesterone ratio, are related to the duration and amplitude of electrical bursts in myometrial cells (Fig. 1). However, in their study, the levels of estrogen and progesterone may merely be a reflection of the stage of the cycle, without proving the role of estrogens and progesterone. In our studies [8], we have observed that sows with a longer duration of estrus (3 versus 2 days) maintained the high level of uterine activity during estrus for a longer period of time. For sows with a longer duration of estrus, the decline in peripheral plasma estrogen levels occurs later relative to the onset of estrus. Variation between sows in the observed myometrial activity may also be caused by variation within sows throughout the day. In a study with mini-pigs [4], myometrial electrical activity showed a rythmic fluctuation throughout the day during proestrus. In a study with Landrace sows [13], the frequency of contractions was constant throughout the day during estrus. If variation throughout the day exists in sows, it might be regulated by varying levels of estrogens, oxytocin, and prostaglandins. Both oxytocin and prostaglandins are known agonists of myometrial activity. In the ewe [14], for example, levels of oxytocin have been shown to fluctuate during estrus. Both estradiol and oxytocin have been shown to affect the
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Fig. 1. Plasma level of estradiol-17b on the first 2 days of estrus, in relation to the duration of electrical bursts recorded in an electromyogram. Different symbols represent different sows, on day 1 or day 2 of estrus. After Hoang-Vu [13].
release of prostaglandin by the uterus [15]. Fluctuation in estradiol and oxytocin may affect uterine activity directly, but also indirectly through the release of prostaglandin. Information on the diurnal variation in oxytocin and prostaglandin levels in pigs during estrus is lacking. One study [16] reports low but fluctuating levels of prostaglandin during estrus in sows. More study is needed to investigate the dynamics of these hormones and the relation with uterine activity during estrus in the pig. A more recently described candidate for regulation of uterine activity is LH. LH/hCG-receptors are present in the myometrium during estrus [17], and hCG has been shown to suppress myometrial activity [18]. Whether the elevated levels of LH during the pre-ovulatory period have a function in the regulation of myometrial activity is still not clear. Obviously, myometrial activity is not only regulated by levels of circulating hormones but also by the sensitivity to these hormones. Estradiol, progesterone, and oxytocin receptor concentrations [19,20] are known to be regulated by estradiol and progesterone (up-regulation by estradiol and down-regulation by progesterone). Sensitivity to oxytocin, for example, increases around estrus ([21,22], sows) but can also differ between animals ([23,24], mares). Another factor that affects myometrial activity is parity. Combined data from several experiments (Langendijk et al., unpublished results) show that, on average, the frequency of contractions during estrus is higher in first parity sows (25/h) than in second and older parity sows (19 and 18/h). The amplitude is 59 mm Hg in primiparous sows and 45 and 51 mm Hg in second and older parity sows. A question that remains to be answered is whether cyclic sows, as used in these studies, have the same level of uterine activity as post-weaning sows. In summary, the variation in spontaneous myometrial activity around estrus is probably a reflection of altering levels of progesterone and estrogens and their receptors. Observed differences between sows are mainly due to sow-dependent factors like the duration of estrus and parity.
3. Male sexual stimuli and uterine activity During and around mating, several sexual stimuli are involved that may be important for their effect on uterine activity. These stimuli can be divided into sensory stimuli, i.e. tactile,
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Fig. 2. Change in frequency of contractions (mean S.E.M.) after treatment with oxytocin, for sows that had either a below average or above average frequency of contractions before treatment. Average frequency of contractions before treatment was 19/h. Sows were either treated with 5 IU oxytocin i.m. (n = 22) or 2 IU oxytocin i.v. (n = 6). There was no difference in myometrial response between the two routes of administration and therefore the data were pooled. (*) Significant difference (P < 0.05) between the two groups.
olfactory, visual and auditory stimuli, on the one hand and seminal plasma-related stimuli on the other. The presence of the boar as such has been shown to increase plasma oxytocin levels dramatically in the sow, and to increase uterine actvity as well [25]. Interestingly, in our work [26], the increase in uterine activity was only observed in sows that had a belowaverage frequency of uterine contractions. The same phenomenon was observed when oxytocin was injected i.m.; sows with a below-average frequency of uterine contractions showed a greater increase in uterine activity (Fig. 2). The increase in uterine activity in the presence of a boar was not related to the magnitude of oxytocin release. The difference between sows in the effect of boar presence or oxytocin injection could have been caused by differences in the sensitivity to oxytocin due to differences in receptor concentrations, but this was not studied. The effects of the components of boar presence or mating have hardly ever been studied separately. Olfactory stimulation with 5a-androstenone can cause a release of oxytocin with the same magnitude as observed during mating, and can increase uterine activity [27,28]. However, the effect of 5a-androstenone on oxytocin release is not unambiguous: Langendijk et al. [26] describe a study in which the spraying of 5a-androstenone did not induce any release of oxytocin and hardly affected myometrial activity at all. In that study, sows were accustomed to regular boar contact (twice a day). Habituation to regular boar contact may reduce responsiveness of sows to the components of boar stimuli, such as boar odor. Whether this is an explanation for the total lack of an olfactory effect on oxytocin release in our study is not clear. Tactile stimulation of the back and the flanks of a sow, in the absence of a boar, does not induce oxytocin release and hardly has any effect on uterine activity [26], even though it evokes receptive behavior. The effects of tactile stimulation of the cervix on myometrial activity have been studied by several authors. Intromission of a normal catheter or manipulation of a transcervical catheter for uterine pressure recording
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does not induce release of oxytocin [25,26]. In a study by Bower [1], sensory stimuli such as massage of the vulva and clitoris, mounting of a boar and intromission of a catheter did not affect uterine activity. However, in his study, observations continued for only about 10 min after a particular treatment, which may be too short to assess the effects reliably. Zerobin [29] found a positive effect on uterine activity of the mere insertion of an insemination catheter, and Pitjkanen and Prokofjev [30] observed enhancement of uterine activity after electrical stimulation of the cervix. Insemination with a given volume of saline or semen extender stimulates contractility ([6]:100 ml; [1]: volume not described; Langendijk et al., unpublished results: 80 ml), but these effects can be ascribed to both the volume of the infused fluid and stimulation of the cervix with the insemination pipette. Intrauterine infusion with 80 ml of saline via a thin intrauterine catheter used for intraluminal pressure recording and thus avoiding cervical stimulation does not enhance uterine activity [8]. All of these studies were performed using a relatively low number of sows. Taken together, however, it seems that the insertion of an insemination catheter, and not the infusion of the volume of fluid associated with insemination, enhances uterine activity. Stimulation of the cervix probably stimulates uterine activity through an adrenergic or cholinergic pathway, as oxytocin is not released. Both adrenergic and cholinergic receptors have been detected in the myometrium, with adrenergic receptors dominating in the longitudinal muscle layer and cholinergic receptors dominating in the circular muscle layer [31]. Stimulation of cholinergic and a-adrenergic receptors initiates contractility, whereas stimulation of b-adrenergic receptors suppresses contractility. It is still not clear which receptor type is predominantly activated by cervical stimulation, but based on the studies mentioned above, a-adrenergic and/or cholinergic stimulation seems to dominate. The effect of excitation of the myometrium, both through the release of oxytocin and through neurogenic stimulation, is maintained for a considerable period of time. Uterine activity remained elevated in the period of recording (ca. 1 h) following presentation of a boar [26] or boar odor (40 min, [28]). Hoang-Vu [13] recorded for a longer period of time and found that the effect of cervical stimulation during insemination lasted for a few hours after insemination. In contrast to the situation for sensory stimulation, there is more convincing evidence for the effect of seminal plasma on uterine activity. Seminal plasma has been shown to stimulate uterine motility in vitro [32]. The reason for this is most likely the estrogen content of seminal plasma. A boar ejaculate can contain up to ca. 12 mg of estrogens, although the level varies between boars and between seasons [33]. Roughly half of the estrogens in an ejaculate are conjugated to the sperm cells, which are likely to act as carrier proteins. After insemination, the estrogens in the ejaculate cause an immediate release of prostaglandin by the endometrium [33]. Intrauterine infusion of both estrogens [33] and prostaglandin [8,34] has been shown to increase uterine motility in sows. The effect of infusion with a physiological dose of estrogens (11.5 mg) on uterine motility is maintained for a few hours [33]. Mating with a boar probably results in an even more prolonged effect on uterine motility because of a more prolonged release of estrogens that are conjugated to sperm cells [33]. In summary, spontaneous myometrial activity during estrus can be influenced by boar stimuli. The presence of the boar clearly induces a release of oxytocin and stimulates myometrial activity, the latter predominantly in sows with relatively low uterine activity. The effects of the components of boar stimuli, such as tactile stimuli and olfactory stimuli,
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are less clear. Cervical stimulation during insemination seems to stimulate uterine activity, but not through the release of oxytocin. Estrogens in seminal plasma have a clear effect on uterine activity through the release of prostaglandin.
4. Uterine activity and the concept of sperm transport During both natural mating and AI, a considerable volume (80–300 ml) is deposited in the genital tract. This volume is functional, because a decrease of the inseminated volume (to 20 ml) reduces the fertilization rate [35]. The large volume probably flushes sperm cells from the cervix into the uterine horns, and prevents the sperm cells from being retained in the cervical folds. For transport and distribution of sperm cells within the remaining part of the tract, a large volume is not necessary since a good fertilization rate can be achieved even with small volumes (less than 10 ml) of semen deposited unilaterally, deep in the uterus [36,37]. Nevertheless, a larger volume may still accelerate the transport of sperm cells to the utero-tubal junction, thus reducing the transit time through the uterine horns. This could be particularly important when insemination takes place long before ovulation, because phagocytosis in the uterine horns reduces the number of sperm cells surviving there [38]. After semen deposition, sperm cells have to be transported to the tubal ends of the uterine horns. Within minutes after insemination, fertile sperm cells can be found in the oviducts [39], indicating that this process can be very fast. The most caudal portions of the uterine horns protrude downwards from the cervix, into the abdominal cavity. The flow of semen from its container (in the case of AI) into the most caudal portion of the uterine horns therefore probably depends on gravity and on relaxation of the myometrium. During estrus, the luminal pressure in the uterus consists of a baseline pressure in the relaxed state, with periodically superimposed increases in pressure due to contractions. A reduction in pressure (relative to the pressure outside the sow) causing suction has never been observed in our studies [8]. Therefore, the common assumption that semen is ‘sucked’ from the AI vial into the genital tract is probably wrong. In contrast, stimulation of contractility can increase the time needed for uptake of semen during insemination [40]. Huhn et al. [41] observed a decrease in pregnancy rate with increasing time needed for insemination. Stimulation of contractions can also increase the reflux of semen [40]. Zerobin and Spo¨ rri [3] observed that contractions in the caudal part of the uterus obstructed infusion of semen. Willenburg et al. [42] also observed an increased amount of reflux during AI when prostaglandin was added to the inseminate. However, in a different study [43], they observed that the presence of a boar reduced the amount of reflux during AI. An increased frequency of contractions probably delays the influx of semen from the AI catheter into the most caudal part of the genital tract, and even increases the risk of reflux, although available data are not totally consistent on this matter. A large amount of reflux during insemination reduces the chance of fertilization [40,44]. Beyond the most caudal portion, the uterine horns curl upwards and end at the utero-tubal junction, near the ovaries. Transport of sperm cells through the remainder of the horns, and distribution of semen over the two horns in the case of unilateral deposition of semen, depends on uterine contractions. Additionally, transfer of sperm cells into the oviducts may in part be promoted by uterine contractions. Shortly after insemination, Baker and Degen [39]
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observed fluid flowing in a pulsatile manner from a uterine cannula positioned near the oviducts. Fluid also appeared in cannulas inserted in the oviducts. Therefore, uterine contractions seem to contribute to the transfer of sperm cell-containing fluid into the oviducts. The effects of suppressing uterine contractility have hardly ever been studied in sows. In luteal sows (day 7), which show no or hardly any uterine activity, inseminated sperm cells can reach the oviducts [45]. In an experiment by Langendijk et al. [40], considerable reduction of uterine activity by infusion of a b-adrenergic agent before insemination reduced the fertilization rate and the number of sperm cells at the site of fertilization. In the same study, suppression of uterine activity increased the number of sperm cells recovered from the uterine horns 12 h after AI, but the number of sperm cells recovered from the oviducts was decreased relative to the uterine horns. It is likely that reduction of uterine contractility prolongs the transit phase in the uterus, reducing sperm quality and thus reducing the ability of sperm cells to enter the oviducts and to fertilize. This is particularly important when insemination takes place long (>24 h) before ovulation. From the work of First et al. [45] it can be concluded that by 24 h after insemination, the sperm cells in the uterine horns are no longer motile, and probably hardly any sperm cells from the uterus enter the oviducts after this time. On the other hand, 16–18 h after insemination, sperm cells that are still capable of in vitro fertilization can be retrieved from the uterine horns (Flowers, personal communication). Further, improving the survival of sperm cells by adding a phagocytosis inhibitor to the insemination dose [46] increases the number of accessory sperm cells even when inseminating 26 h before ovulation. More studies are needed to investigate how sperm cells survive in the uterus, whether sperm cells can still enter the oviducts and fertilize in vivo after long-term incubation in the uterus and how uterine contractions affect transit through the uterine horns. Stimulation of uterine contractility in sows has been studied far more extensively than suppression. Waberski et al. [47] observed an increase in the number of accessory sperm cells in the zona pellucida of 3- to 4-day-old embryos when seminal plasma was infused before insemination. The fertilization rate was not affected. Infusion of seminal plasma in one horn promoted the transport of sperm cells into the ipsilateral oviduct during 1–6 h after insemination [48]. Intravenous injection of a supraphysiological dose of 10 IU oxytocin, shortly after insemination with a low volume of semen, improved the percentage of fertilized ova from 58 to 72% [49]. Probably, stimulation of uterine contractility can improve sperm transport and/or fertilization, but will also increase reflux. This is particularly important in unfavorable situations, i.e. with a low number of sperm cells, a low volume of inseminate, a long interval from insemination to ovulation, etc. Pena et al. [50,51], for example, showed that hormonal stimulation of uterine contractions at the time of insemination can improve fertilization during the low-fertility season. However, stimulation of uterine contractility may also impede sperm transport to the oviducts and fertilization, probably depending on the extent of stimulation. Intrauterine infusion of 1 mg of cloprostenol before insemination, a dose sufficient to double the frequency of uterine contractions [8], increased the reflux of semen during insemination, reduced the number of sperm cells in the oviducts, and reduced the fertilization rate ([40], Table 2). Addition of 10 IU of oxytocin to the semen dose reduced the percentage of fertilized ova, whereas i.v. injection of the same dose 5 min after insemination improved the fertilization rate [49].
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Table 2 Reflux of semen during insemination as affected by stimulation (cloprostenol, 1 mg in the AI dose) or suppression (clenbuterol-HCl, 0.60 mg in the AI dose) of uterine contractions, and the effect on fertilization rate Amount of reflux No
Small
Large
Treatment Cloprostenol Control Clenbuterol
4 12 16
9 3 2
6 2 0
Fertilization ratea (%) 0–33 33–67 67–100
13 4 13
6 4 4
7 0 1
Numbers represent sows in each category. a Fertilization rate: percentage of fertilized oocytes at 5 days after ovulation.
The magnitude of uterine contractility after stimulation and probably also the timing of stimulation relative to the time of insemination affect the reflux of semen. This phenomenon may explain the inconsistency found in the effect of hormonal stimulation of uterine contractility on reproductive performance in field trials [52]. Introduction of a boar probably stimulates uterine contractility to an appropriate level, as this increases uterine activity only in sows with low spontaneous uterine activity ([26], Fig. 3). In summary, a
Fig. 3. Change in frequency of uterine contractions (mean S.E.M.) after one of three different stimuli, for sows that had either a below average or above average frequency of contractions before treatment. Average frequency of contractions before treatment was 25/h. The three different stimuli were: (1) BPT (n = 15): back pressure test, i.e. manual stimulation of the back and the flanks of a sow; (2) SPRAY (n = 12): a BPT in combination with boar pheromone spray; (3) BOAR (n = 15): a BPT in the presence of a boar. (*) Significant difference between different stimuli (P < 0.05). In the sows with an above average frequency of contractions before treatment, the treatments had no effect. In the sows with a below average frequency of contractions before treatment, all three stimuli increased frequency significantly (P < 0.05), but the effect of BOAR was greater than the other two stimuli (P < 0.05).
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certain degree of uterine contractility is probably important for rapid transport of sperm cells to the relatively safe oviducts, especially in situations that are unfavorable for fertilization. Stimulation of contractility can, to a certain extent, be favorable for the transport of sperm cells and fertilization. A (too) high level of uterine activity probably reduces uptake of semen by the uterine horns and increases the risk of reflux. An important issue in the concept of sperm transport is the direction in which uterine contractions are propagated along the uterine horns. Waves of uterine contraction are thought to originate predominantly at the tubal and cervical ends of the uterine horns, these being the sites with the most pacemaker activity, at least during parturition [4]. Tubocervical directed contractions are thought to be important to expel seminal plasma after mating [38], but are probably also important in the distribution of semen over the two horns. After unilateral insemination, fertilization in the contralateral horn is similar to the fertilization rate in the ipsilateral horn, even with low sperm numbers (5 107) and a small volume (10 ml) [37]. Recently, however, evidence has also been provided for transport of sperm through the abdominal cavity to the contralateral oviduct (E. Martinez, personal communications). Cervico-tubal contractions are thought to be important in the transport of semen to the sperm reservoirs. Propagation of contractions depends on communication between myometrial cells, which is increased during estrus, when the number of gap junctions is increased due to the high level of estrogens [10,11]. We hypothesise that observations on the direction of uterine contractions probably depend on the site of measurement. In the tubal end of the horn, for example, cervically directed contractions probably dominate, because this site is located nearest to the tubal pacemaker zone. Contractions originating cervically are less likely to be propagated all the way to this site than contractions originating tubally. Studies on the direction of uterine contractions report tubo-cervical, cervico-tubal, and undirected contractions [2,3,5]. Another factor complicating studies on the direction of uterine contractions is the interpretation of pressure recordings. When several contractions follow closely in time, it is difficult to identify in what direction the contractions pass the recording sensors Nevertheless, reports on the direction of contractility claim that the direction of contractions is a coordinated process, which depends on the stage of estrus [2,3,5] and can be affected by stimuli involved in mating [3]. In summary, there is evidence for tubo-cervical, cervico-tubal, and undirected contractility, which are observed simultaneously throughout estrus. More information is needed to elucidate the coordination of contractility during different stages of estrus and the effects of external stimuli.
5. Conclusions Spontaneous uterine activity increases during estrus and has a function in the transport of sperm cells to the oviducts. This is particularly important when other factors around insemination, like semen dose, time of insemination relative to ovulation, etc., limit the number of sperm cells that survive until the moment of fertilization. The variation in uterine activity between sows is consistent throughout estrus and may contribute to the variation in fertilization results between sows. Stimulation of uterine contractility around insemination can improve sperm transport to the oviducts, but can also affect sperm
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transport and fertilization negatively. The latter occurs when contractility is stimulated to such an extent that the uptake of semen by the genital tract is obstructed and reflux of semen is increased. Introduction of a boar during insemination is probably an adequate way of stimulating contractility because oxytocin release is effectively induced in all sows and because boar presence selectively stimulates contractility in those sows with low spontaneous uterine activity. Future research should focus on the direction of uterine contractions and its regulation, and on the importance of separate components of the complex of boar stimuli for uterine contractility.
References [1] Bower RE. Factors affecting myometrial activity in the pig. Thesis, University of Minnesota; 1974. [2] Scheerboom JE, Van Adrichem PW, Taverne MAM. Uterine motility of the sow during the estrous cycle and early pregnancy. Vet Res Commun 1987;11:253–69. [3] Zerobin K, Spo¨ rri H. Motility of the bovine and porcine uterus and fallopian tube. Adv Vet Sci 1972;16: 303–54. [4] Taverne MAM. Myometrial activity during pregnancy and parturition in the pig. In: Cole DJA, Foxcroft GR, editors. Control of pig reproduction. London: Butterworths; 1982. p. 419–36. [5] Bru¨ ssow KP, Kurth J, Blo¨ dow G. Electrophysiological studies into uterus motoricity of gilts during estrus, following synchronization of ovulation. Arch Exp Vet Med 1988;42:933–43. [6] Claus R, Ellendorff F, Hoang-Vu C. Spontaneous electromyographic activity throughout the cycle in the sow and its change by intrauterine estrogen infusion during estrus. J Reprod Fertil 1989;87:543–51. [7] Von Do¨ cke F, Worch H. Investigations into uterine motility and mating reactions of sows. Zuchthygiene 1963;7:169–78. [8] Langendijk P, Bouwman EG, Soede NM, Taverne MAM. Kemp B. Myometrial activity around estrus in sows: spontaneous activity and effects of estrogens, cloprostenol, seminal plasma and clenbuterol. Theriogenology 2002;57:1563–77. [9] Hazeleger W, Kemp B. Farrowing rate and litter size after transcervical embryo transfer in sows. Reprod Dom Anim 1994;29:481–7. [10] Verhoeff A, Garfield RE, Ramondt J, Wallenburg HC. Electrical and mechanical uterine activity and gap junctions in estrogen-treated oophorectomized sheep. Am J Obstet Gynecol 1986;155:1192–6. [11] Michael CA, Schofield BM. The influence of the ovarian hormones on the actomyosin content and the development of tension in uterine muscle. J Endocrinol 1969;44:501–11. [12] Finn CA, Porter DG. The uterus. London: Elek Science; 1975, p. 149–65. [13] Hoang-Vu C. Elektrische Aktivita¨ t des Myometriums beim Schwein wa¨ hrend des Zyklus und ihre ¨ strogenen in das Uteruslumen am Tag der Rausche. Thesis, Hohenheim Beeinflussung durch Infusion von O University; 1987. [14] Gilbert CL, Jenkins K, Wathes DC. Pulsatile release of oxytocin into the circulation of the ewe during oestrus, mating and the early luteal phase. J Reprod Fertil 1991;91:337–46. [15] Edgerton LA, Kaminski MA, Silvia WJ. Effects of progesterone and estradiol on uterine secretion of prostaglandin F2a in response to oxytocin in ovariectomised sows. Biol Reprod 2000;62:365–9. [16] Shille VM, Karlbom I, Einarsson S, Larsson K, Kindahl H, Edqvist LE. Concentrations of progesterone and 15-keto-13, 14-dihydroprostaglandin F2a in peripheral plasma during the estrous cycle and early pregnancy in gilts. Zentralbl Veterinarmed A 1979;26:169–81. [17] Ziecik AJ, Derecka-Reszka KA, Rzucidlo SJ. Extragonadal gonadotropin receptors, their distribution and function. J Physiol Pharmacol 1992;43(Suppl 1):33–49. [18] Flowers B, Ziecik AJ, Caruolo EV. Effects of human chorionic gonadotropin on contractile activity of steroid-primed pig myometrium. J Reprod Fertil 1991;92:425–32. [19] Thilander G, Eriksson H, Edqvist LE, Rodriguez-Martinez H. Progesterone and estradiol-17 beta receptors in the porcine myometrium during the estrous cycle. Zentralbl Veterinarmed A 1990;37:321–8.
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[44] Steverink DWB, Soede NM, Bouwman EG, Kemp B. Semen backflow after insemination and its effect on fertilisation in sows. Anim Reprod Sci 1998;54:109–19. [45] First NL, Short RE, Peters JB, Stratman FW. Transport of boar spermatozoa in estrual and luteal sows. J Anim Sci 1968;27:1032–6. [46] Woelders H, Matthijs JJ, Bouwman EG, Soede NM. Caffeine plus Ca2+ reduces uterine leukocyte recruitment and sperm phagocytosis and improves fertility in pig AI. In: Proceedings of the 14th International Congress on Animal Reproduction, Stockholm 2000. Proc 2, 94 (Abstract 15:20). [47] Waberski D. Effect of a transcervical infusion of seminal plasma prior to insemination on the fertilising competence of low numbers of boar spermatozoa at controlled AI-ovulation intervals. Anim Reprod Sci 1996;44:165–73. [48] Viring S, Einarsson S. Influence of boar seminal plasma on the distribution of spermatozoa in the genital tract of gilts. Acta Vet Scand 1980;21:598–606. [49] Stratman FW, Self HL, Smith VR. The effect of oxytocin on the fertility in gilts artificially inseminated with a low sperm concentration and semen volume. J Anim Sci 1959;18:634–40. [50] Pena FJ, Dominguez JC, Pelaez J, Alegre B. Intrauterine infusion of PGF(2alpha) at insemination enhances reproductive performance of sows during the low fertility season. Vet J 2000;159:259–61. [51] Pena FJ, Dominguez JC, Carbajo M, Anel L, Alegre B. Treatment of swine summer infertility syndrome by means of oxytocin under field conditions. Theriogenology 1998;49:829–36. [52] Levis DG. The effect of oxytocin at the time of insemination on reproductive performance—a review. Nebraska Swine Report 2000, pp. 11–17.
Uterine activity, sperm transport, and the role of boar stimuli around insemination in sows P. Langendijk*, N.M. Soede, B. Kemp Department of Animal Science, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands
Abstract This paper describes changes in spontaneous myometrial activity around estrus, factors that affect myometrial activity, and the possible role of uterine contractions in the process of (artificial) insemination, sperm transport and fertilization. Myometrial activity in the sow increases during estrus. The activity is myogenic in origin, but several factors have been shown to affect myometrial activity. Natural mating stimulates uterine contractions through several mechanisms. The presence of a boar, rather than the act of mating, induces central oxytocin release in the sow and thus increases uterine activity. Estrogens in the ejaculate of a boar can trigger prostaglandin release by the endometrium and thus increase uterine activity. Tactile stimulation of the genital tract (cervix) or tactile stimulation of the back and flanks of the sow during artificial insemination does not cause a release of oxytocin. There is hardly any evidence for the effects of these latter stimuli on uterine activity, and if they are present at all, the effects are very small. Evidence for the effects of synthetic boar odor on oxytocin release and/or uterine activity is inconsistent. The mere presence of a boar during insemination, in contrast, clearly stimulates uterine activity through the release of oxytocin. Hormonal stimulation (intrauterine) of uterine activity with estrogens, prostaglandins, or oxytocins before, during or after insemination generally improves fertilization rate, especially in situations with reduced fertility. Therefore, uterine contractions are believed to play an important role in the transport of sperm cells to the oviducts after insemination. Whether uterine contractions are absolutely necessary for sperm transport through the uterine horns, however, is not clear. Intensive stimulation of uterine contractions using hormones can also reduce the fertilization rate, probably by increasing the reflux of sperm cells during insemination. In this respect, the presence of a boar during
* Corresponding author. Tel.: +31 317 483197; fax: +31 317 485006. E-mail address: [email protected] (P. Langendijk). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.09.027
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AI seems more adequate, as only sows with a low level of uterine activity show an increase in uterine activity in response to this stimulus. # 2004 Elsevier Inc. All rights reserved. Keywords: Uterine contractions; Sperm transport; Boar stimuli; Fertilization; Insemination
1. Introduction During mating and artificial insemination in sows, semen is deposited intra-cervically. However, due to the volume, the bulk of the semen is flushed directly into the lumen of the uterine body, immediately before the cervix. Therefore, semen deposition is often referred to as intrauterine. From the site of deposition, sperm cells must be distributed over both horns and transported to the tubal end of the horns. There, a relatively small number populates the utero-tubal junction and the first part of the oviduct, which serve as a sperm reservoir. The transport of sperm cells through the uterine horns is believed to be a passive process, in which intrinsic sperm cell motility plays no part. This passive process probably depends on the flow of sperm cell-containing fluids in the uterine lumen, driven by gravitational force and uterine contractility. In the present paper, the available literature on uterine activity around estrus in sows is reviewed, and the role of uterine activity in the transport of sperm cells and the effects of boar stimuli are discussed.
2. Spontaneous uterine activity Uterine activity around estrus in sows has been studied using several invasive methods (Table 1), either by applying strain gauges to the uterine wall to record the stretch exerted by the uterine musculature [1,2] or by applying electrodes to record electromyographic activity [3–6]. A drawback of both strain gauges and electromyography is that these methods do not directly record pressure changes in the uterine lumen, since electrical activity does not necessarily induce muscular contraction. This translation depends on the sensitivity of the myometrium to electrical triggering, which in turn depends on the physiological status of the myometrium [2]. Several types of electrical activity, as defined by Scheerboom et al. [2], all coincide with myometrial contractions as recorded with strain gauges. However, it is not clear from the study by Scheerboom et al. [2] how electrical activity correlates quantitatively with myometrial contraction. Intraluminal pressure changes are a more direct indication of muscular contraction, and intraluminal pressure changes after all are the driving force for transport of fluids (e.g. semen) from one place to the other. Do¨ cke and Worch [7] recorded uterine intraluminal pressure using a fluid-filled balloon inserted into the cervical end of the uterus. There are no other reports on the use of non-invasive techniques to record intraluminal pressure in sows. Over the past few years, we have developed a non-invasive technique [8] by adapting a device developed for nonsurgical embryo transfer in pigs [9]. With this device it is possible to pass the cervix and introduce an open-ended, fluid-filled catheter into the caudal end of the uterine horns to
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Table 1 Myometrial activity during and around estrus, as reported by different authors, using different methods Author and method
Parameter a
Diestrus
Proestrus
Estrus
Scheerboom (1987) EMGb
Frequency of MC Amplitude
4/h 115 mV
14/h 227 mV
10/h 870 mV
Claus et al. (1989) EMG
Frequency of burstsc Duration of bursts Amplitude Luteal phase activity below detection level
90/h 2s 180 mV
17/h 22 s 1720 mV
20/h 32 s 2040 mV
Brussow et al. (1988) EMG
Frequency of APd Relative duratione
8/h 22%
29/h 25%
Bower (1974) Strain gauges
Frequency of contractions Force of stretch
0/h 0g
8/h 14 g
15/h 15 g
Zerobin and Spo¨ rri (1972)f Intraluminal pressure
Frequency of contractions Amplitude of contractions Duration of contractions
<8 mmHg
60/h <15 mmHg 25 s
21–30/h 35–45 mmHg Maximum 60 s
Langendijk et al. (2002a) Intraluminal pressure
Frequency of contractions Amplitude of contractions Duration of contractions
15/h 20 mm Hg 60 s
22/h 40 mm Hg 60 s
a b c d e f
MC = myoelectric Complex. EMG = electromyography. Bursts: bursts of electrical activity. AP = action potentials. Relative duration: duration of electrical activity, relative to measuring time. For Zerobin and Spo¨ rri, approximate figures are given because of a lack of quantitative information.
record intraluminal uterine pressure. With this technique, we have studied spontaneous myometrial activity around estrus. Two to four days before estrus, myometrial activity is low in terms of frequency and amplitude of contractions [8]. A percentage of sows (around 50%) does not show any contractility at all around this time, and the sows that do show contractility tend to have a low frequency and amplitude of contractions, compared to the situation during estrus (Table 1). Approaching estrus, the percentage of sows showing myometrial activity increases, and the frequency and amplitude of contractions increases too. During estrus, all sows show myometrial activity and the frequency and amplitude of contractions are maximal. After estrus, uterine activity decreases again. Using the non-invasive technique, data could be collected in a relatively large number of sows [8], which enabled us to study the variation between sows. The variation between sows during estrus is huge: the frequency of contractions ranges from 6 to 40/h and average amplitude per sow ranges from 16 to 57 mm Hg. Nevertheless, the difference between sows is consistent over the days around estrus, i.e., sows with a relatively high level of uterine activity during the days before estrus also show a relatively high level of uterine activity during estrus. This indicates a sow-dependent variation throughout the estrous cycle. Variation between sows over cycles also exists: for cyclic sows in which more than one estrous cycle was monitored, the variation between sows explained 60% of the total variation (cycle
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variation + sow variation) in contraction frequency during estrus (Langendijk et al., unpublished results). Variation in uterine activity during the estrous cycle has also been observed by other authors. The force of contraction increases during estrus, regardless of what method is used [1–3,5,6], but not all authors report an increase in the frequency of contractions during estrus (Table 1). However, quantitative comparisons between studies are difficult because the site of recording, the method of recording (electrical activity, muscle stretch and intraluminal pressure), and the definition of parameters differ between studies. With electromyographic observations, a lower frequency of electrical activity is sometimes reported during estrus than outside of estrus [2,6]. However, the lower frequency of electrical activity during estrus does not necessarily correlate with lower contractile activity (see data of Scheerboom et al. [2] on electromyography and strain gauges). During estrus, the amplitude and duration of electrical bursts are more pronounced [6] and the myometrium is more sensitive to electrical input. Thus, a lower frequency of electrical bursts (that are more pronounced) during estrus can still result in a higher frequency of contractions. Nevertheless, Zerobin and Spo¨ rri [3] also observed a decrease in the frequency of contractions during estrus, and they used intraluminal catheters. However, the contractions during estrus (low frequency, high amplitude) were of much higher amplitude (35–45 mm Hg) compared to the contractions outside estrus (high frequency, low amplitude; up to 3 mm Hg), and with different criteria (a higher threshold to accept a pressure fluctuation as a contraction) a lower frequency of contractions would probably have been recorded outside estrus. In conclusion, the amplitude of contractions is increased during estrus and the frequency of contractions is highly dependent on the criteria used to define contractility. However, ‘functional’ uterine activity, in terms of sperm transport, is probably increased during estrus. The variation in myometrial activity throughout the estrous cycle and between sows probably depends mainly on the tissue and plasma levels of estrogen and progesterone. Progesterone inhibits and estrogen increases uterine activity. From studies on other species than the pig, it appears that estrogen increases gap junctional coupling between myometrial cells [10], the myosin content of myometrial cells [11], and the pacemaker activity of myometrial cells [12]. Data from Hoang-Vu [13] indicate that estrogen levels, but also the estrogen/progesterone ratio, are related to the duration and amplitude of electrical bursts in myometrial cells (Fig. 1). However, in their study, the levels of estrogen and progesterone may merely be a reflection of the stage of the cycle, without proving the role of estrogens and progesterone. In our studies [8], we have observed that sows with a longer duration of estrus (3 versus 2 days) maintained the high level of uterine activity during estrus for a longer period of time. For sows with a longer duration of estrus, the decline in peripheral plasma estrogen levels occurs later relative to the onset of estrus. Variation between sows in the observed myometrial activity may also be caused by variation within sows throughout the day. In a study with mini-pigs [4], myometrial electrical activity showed a rythmic fluctuation throughout the day during proestrus. In a study with Landrace sows [13], the frequency of contractions was constant throughout the day during estrus. If variation throughout the day exists in sows, it might be regulated by varying levels of estrogens, oxytocin, and prostaglandins. Both oxytocin and prostaglandins are known agonists of myometrial activity. In the ewe [14], for example, levels of oxytocin have been shown to fluctuate during estrus. Both estradiol and oxytocin have been shown to affect the
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Fig. 1. Plasma level of estradiol-17b on the first 2 days of estrus, in relation to the duration of electrical bursts recorded in an electromyogram. Different symbols represent different sows, on day 1 or day 2 of estrus. After Hoang-Vu [13].
release of prostaglandin by the uterus [15]. Fluctuation in estradiol and oxytocin may affect uterine activity directly, but also indirectly through the release of prostaglandin. Information on the diurnal variation in oxytocin and prostaglandin levels in pigs during estrus is lacking. One study [16] reports low but fluctuating levels of prostaglandin during estrus in sows. More study is needed to investigate the dynamics of these hormones and the relation with uterine activity during estrus in the pig. A more recently described candidate for regulation of uterine activity is LH. LH/hCG-receptors are present in the myometrium during estrus [17], and hCG has been shown to suppress myometrial activity [18]. Whether the elevated levels of LH during the pre-ovulatory period have a function in the regulation of myometrial activity is still not clear. Obviously, myometrial activity is not only regulated by levels of circulating hormones but also by the sensitivity to these hormones. Estradiol, progesterone, and oxytocin receptor concentrations [19,20] are known to be regulated by estradiol and progesterone (up-regulation by estradiol and down-regulation by progesterone). Sensitivity to oxytocin, for example, increases around estrus ([21,22], sows) but can also differ between animals ([23,24], mares). Another factor that affects myometrial activity is parity. Combined data from several experiments (Langendijk et al., unpublished results) show that, on average, the frequency of contractions during estrus is higher in first parity sows (25/h) than in second and older parity sows (19 and 18/h). The amplitude is 59 mm Hg in primiparous sows and 45 and 51 mm Hg in second and older parity sows. A question that remains to be answered is whether cyclic sows, as used in these studies, have the same level of uterine activity as post-weaning sows. In summary, the variation in spontaneous myometrial activity around estrus is probably a reflection of altering levels of progesterone and estrogens and their receptors. Observed differences between sows are mainly due to sow-dependent factors like the duration of estrus and parity.
3. Male sexual stimuli and uterine activity During and around mating, several sexual stimuli are involved that may be important for their effect on uterine activity. These stimuli can be divided into sensory stimuli, i.e. tactile,
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Fig. 2. Change in frequency of contractions (mean S.E.M.) after treatment with oxytocin, for sows that had either a below average or above average frequency of contractions before treatment. Average frequency of contractions before treatment was 19/h. Sows were either treated with 5 IU oxytocin i.m. (n = 22) or 2 IU oxytocin i.v. (n = 6). There was no difference in myometrial response between the two routes of administration and therefore the data were pooled. (*) Significant difference (P < 0.05) between the two groups.
olfactory, visual and auditory stimuli, on the one hand and seminal plasma-related stimuli on the other. The presence of the boar as such has been shown to increase plasma oxytocin levels dramatically in the sow, and to increase uterine actvity as well [25]. Interestingly, in our work [26], the increase in uterine activity was only observed in sows that had a belowaverage frequency of uterine contractions. The same phenomenon was observed when oxytocin was injected i.m.; sows with a below-average frequency of uterine contractions showed a greater increase in uterine activity (Fig. 2). The increase in uterine activity in the presence of a boar was not related to the magnitude of oxytocin release. The difference between sows in the effect of boar presence or oxytocin injection could have been caused by differences in the sensitivity to oxytocin due to differences in receptor concentrations, but this was not studied. The effects of the components of boar presence or mating have hardly ever been studied separately. Olfactory stimulation with 5a-androstenone can cause a release of oxytocin with the same magnitude as observed during mating, and can increase uterine activity [27,28]. However, the effect of 5a-androstenone on oxytocin release is not unambiguous: Langendijk et al. [26] describe a study in which the spraying of 5a-androstenone did not induce any release of oxytocin and hardly affected myometrial activity at all. In that study, sows were accustomed to regular boar contact (twice a day). Habituation to regular boar contact may reduce responsiveness of sows to the components of boar stimuli, such as boar odor. Whether this is an explanation for the total lack of an olfactory effect on oxytocin release in our study is not clear. Tactile stimulation of the back and the flanks of a sow, in the absence of a boar, does not induce oxytocin release and hardly has any effect on uterine activity [26], even though it evokes receptive behavior. The effects of tactile stimulation of the cervix on myometrial activity have been studied by several authors. Intromission of a normal catheter or manipulation of a transcervical catheter for uterine pressure recording
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does not induce release of oxytocin [25,26]. In a study by Bower [1], sensory stimuli such as massage of the vulva and clitoris, mounting of a boar and intromission of a catheter did not affect uterine activity. However, in his study, observations continued for only about 10 min after a particular treatment, which may be too short to assess the effects reliably. Zerobin [29] found a positive effect on uterine activity of the mere insertion of an insemination catheter, and Pitjkanen and Prokofjev [30] observed enhancement of uterine activity after electrical stimulation of the cervix. Insemination with a given volume of saline or semen extender stimulates contractility ([6]:100 ml; [1]: volume not described; Langendijk et al., unpublished results: 80 ml), but these effects can be ascribed to both the volume of the infused fluid and stimulation of the cervix with the insemination pipette. Intrauterine infusion with 80 ml of saline via a thin intrauterine catheter used for intraluminal pressure recording and thus avoiding cervical stimulation does not enhance uterine activity [8]. All of these studies were performed using a relatively low number of sows. Taken together, however, it seems that the insertion of an insemination catheter, and not the infusion of the volume of fluid associated with insemination, enhances uterine activity. Stimulation of the cervix probably stimulates uterine activity through an adrenergic or cholinergic pathway, as oxytocin is not released. Both adrenergic and cholinergic receptors have been detected in the myometrium, with adrenergic receptors dominating in the longitudinal muscle layer and cholinergic receptors dominating in the circular muscle layer [31]. Stimulation of cholinergic and a-adrenergic receptors initiates contractility, whereas stimulation of b-adrenergic receptors suppresses contractility. It is still not clear which receptor type is predominantly activated by cervical stimulation, but based on the studies mentioned above, a-adrenergic and/or cholinergic stimulation seems to dominate. The effect of excitation of the myometrium, both through the release of oxytocin and through neurogenic stimulation, is maintained for a considerable period of time. Uterine activity remained elevated in the period of recording (ca. 1 h) following presentation of a boar [26] or boar odor (40 min, [28]). Hoang-Vu [13] recorded for a longer period of time and found that the effect of cervical stimulation during insemination lasted for a few hours after insemination. In contrast to the situation for sensory stimulation, there is more convincing evidence for the effect of seminal plasma on uterine activity. Seminal plasma has been shown to stimulate uterine motility in vitro [32]. The reason for this is most likely the estrogen content of seminal plasma. A boar ejaculate can contain up to ca. 12 mg of estrogens, although the level varies between boars and between seasons [33]. Roughly half of the estrogens in an ejaculate are conjugated to the sperm cells, which are likely to act as carrier proteins. After insemination, the estrogens in the ejaculate cause an immediate release of prostaglandin by the endometrium [33]. Intrauterine infusion of both estrogens [33] and prostaglandin [8,34] has been shown to increase uterine motility in sows. The effect of infusion with a physiological dose of estrogens (11.5 mg) on uterine motility is maintained for a few hours [33]. Mating with a boar probably results in an even more prolonged effect on uterine motility because of a more prolonged release of estrogens that are conjugated to sperm cells [33]. In summary, spontaneous myometrial activity during estrus can be influenced by boar stimuli. The presence of the boar clearly induces a release of oxytocin and stimulates myometrial activity, the latter predominantly in sows with relatively low uterine activity. The effects of the components of boar stimuli, such as tactile stimuli and olfactory stimuli,
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are less clear. Cervical stimulation during insemination seems to stimulate uterine activity, but not through the release of oxytocin. Estrogens in seminal plasma have a clear effect on uterine activity through the release of prostaglandin.
4. Uterine activity and the concept of sperm transport During both natural mating and AI, a considerable volume (80–300 ml) is deposited in the genital tract. This volume is functional, because a decrease of the inseminated volume (to 20 ml) reduces the fertilization rate [35]. The large volume probably flushes sperm cells from the cervix into the uterine horns, and prevents the sperm cells from being retained in the cervical folds. For transport and distribution of sperm cells within the remaining part of the tract, a large volume is not necessary since a good fertilization rate can be achieved even with small volumes (less than 10 ml) of semen deposited unilaterally, deep in the uterus [36,37]. Nevertheless, a larger volume may still accelerate the transport of sperm cells to the utero-tubal junction, thus reducing the transit time through the uterine horns. This could be particularly important when insemination takes place long before ovulation, because phagocytosis in the uterine horns reduces the number of sperm cells surviving there [38]. After semen deposition, sperm cells have to be transported to the tubal ends of the uterine horns. Within minutes after insemination, fertile sperm cells can be found in the oviducts [39], indicating that this process can be very fast. The most caudal portions of the uterine horns protrude downwards from the cervix, into the abdominal cavity. The flow of semen from its container (in the case of AI) into the most caudal portion of the uterine horns therefore probably depends on gravity and on relaxation of the myometrium. During estrus, the luminal pressure in the uterus consists of a baseline pressure in the relaxed state, with periodically superimposed increases in pressure due to contractions. A reduction in pressure (relative to the pressure outside the sow) causing suction has never been observed in our studies [8]. Therefore, the common assumption that semen is ‘sucked’ from the AI vial into the genital tract is probably wrong. In contrast, stimulation of contractility can increase the time needed for uptake of semen during insemination [40]. Huhn et al. [41] observed a decrease in pregnancy rate with increasing time needed for insemination. Stimulation of contractions can also increase the reflux of semen [40]. Zerobin and Spo¨ rri [3] observed that contractions in the caudal part of the uterus obstructed infusion of semen. Willenburg et al. [42] also observed an increased amount of reflux during AI when prostaglandin was added to the inseminate. However, in a different study [43], they observed that the presence of a boar reduced the amount of reflux during AI. An increased frequency of contractions probably delays the influx of semen from the AI catheter into the most caudal part of the genital tract, and even increases the risk of reflux, although available data are not totally consistent on this matter. A large amount of reflux during insemination reduces the chance of fertilization [40,44]. Beyond the most caudal portion, the uterine horns curl upwards and end at the utero-tubal junction, near the ovaries. Transport of sperm cells through the remainder of the horns, and distribution of semen over the two horns in the case of unilateral deposition of semen, depends on uterine contractions. Additionally, transfer of sperm cells into the oviducts may in part be promoted by uterine contractions. Shortly after insemination, Baker and Degen [39]
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observed fluid flowing in a pulsatile manner from a uterine cannula positioned near the oviducts. Fluid also appeared in cannulas inserted in the oviducts. Therefore, uterine contractions seem to contribute to the transfer of sperm cell-containing fluid into the oviducts. The effects of suppressing uterine contractility have hardly ever been studied in sows. In luteal sows (day 7), which show no or hardly any uterine activity, inseminated sperm cells can reach the oviducts [45]. In an experiment by Langendijk et al. [40], considerable reduction of uterine activity by infusion of a b-adrenergic agent before insemination reduced the fertilization rate and the number of sperm cells at the site of fertilization. In the same study, suppression of uterine activity increased the number of sperm cells recovered from the uterine horns 12 h after AI, but the number of sperm cells recovered from the oviducts was decreased relative to the uterine horns. It is likely that reduction of uterine contractility prolongs the transit phase in the uterus, reducing sperm quality and thus reducing the ability of sperm cells to enter the oviducts and to fertilize. This is particularly important when insemination takes place long (>24 h) before ovulation. From the work of First et al. [45] it can be concluded that by 24 h after insemination, the sperm cells in the uterine horns are no longer motile, and probably hardly any sperm cells from the uterus enter the oviducts after this time. On the other hand, 16–18 h after insemination, sperm cells that are still capable of in vitro fertilization can be retrieved from the uterine horns (Flowers, personal communication). Further, improving the survival of sperm cells by adding a phagocytosis inhibitor to the insemination dose [46] increases the number of accessory sperm cells even when inseminating 26 h before ovulation. More studies are needed to investigate how sperm cells survive in the uterus, whether sperm cells can still enter the oviducts and fertilize in vivo after long-term incubation in the uterus and how uterine contractions affect transit through the uterine horns. Stimulation of uterine contractility in sows has been studied far more extensively than suppression. Waberski et al. [47] observed an increase in the number of accessory sperm cells in the zona pellucida of 3- to 4-day-old embryos when seminal plasma was infused before insemination. The fertilization rate was not affected. Infusion of seminal plasma in one horn promoted the transport of sperm cells into the ipsilateral oviduct during 1–6 h after insemination [48]. Intravenous injection of a supraphysiological dose of 10 IU oxytocin, shortly after insemination with a low volume of semen, improved the percentage of fertilized ova from 58 to 72% [49]. Probably, stimulation of uterine contractility can improve sperm transport and/or fertilization, but will also increase reflux. This is particularly important in unfavorable situations, i.e. with a low number of sperm cells, a low volume of inseminate, a long interval from insemination to ovulation, etc. Pena et al. [50,51], for example, showed that hormonal stimulation of uterine contractions at the time of insemination can improve fertilization during the low-fertility season. However, stimulation of uterine contractility may also impede sperm transport to the oviducts and fertilization, probably depending on the extent of stimulation. Intrauterine infusion of 1 mg of cloprostenol before insemination, a dose sufficient to double the frequency of uterine contractions [8], increased the reflux of semen during insemination, reduced the number of sperm cells in the oviducts, and reduced the fertilization rate ([40], Table 2). Addition of 10 IU of oxytocin to the semen dose reduced the percentage of fertilized ova, whereas i.v. injection of the same dose 5 min after insemination improved the fertilization rate [49].
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Table 2 Reflux of semen during insemination as affected by stimulation (cloprostenol, 1 mg in the AI dose) or suppression (clenbuterol-HCl, 0.60 mg in the AI dose) of uterine contractions, and the effect on fertilization rate Amount of reflux No
Small
Large
Treatment Cloprostenol Control Clenbuterol
4 12 16
9 3 2
6 2 0
Fertilization ratea (%) 0–33 33–67 67–100
13 4 13
6 4 4
7 0 1
Numbers represent sows in each category. a Fertilization rate: percentage of fertilized oocytes at 5 days after ovulation.
The magnitude of uterine contractility after stimulation and probably also the timing of stimulation relative to the time of insemination affect the reflux of semen. This phenomenon may explain the inconsistency found in the effect of hormonal stimulation of uterine contractility on reproductive performance in field trials [52]. Introduction of a boar probably stimulates uterine contractility to an appropriate level, as this increases uterine activity only in sows with low spontaneous uterine activity ([26], Fig. 3). In summary, a
Fig. 3. Change in frequency of uterine contractions (mean S.E.M.) after one of three different stimuli, for sows that had either a below average or above average frequency of contractions before treatment. Average frequency of contractions before treatment was 25/h. The three different stimuli were: (1) BPT (n = 15): back pressure test, i.e. manual stimulation of the back and the flanks of a sow; (2) SPRAY (n = 12): a BPT in combination with boar pheromone spray; (3) BOAR (n = 15): a BPT in the presence of a boar. (*) Significant difference between different stimuli (P < 0.05). In the sows with an above average frequency of contractions before treatment, the treatments had no effect. In the sows with a below average frequency of contractions before treatment, all three stimuli increased frequency significantly (P < 0.05), but the effect of BOAR was greater than the other two stimuli (P < 0.05).
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certain degree of uterine contractility is probably important for rapid transport of sperm cells to the relatively safe oviducts, especially in situations that are unfavorable for fertilization. Stimulation of contractility can, to a certain extent, be favorable for the transport of sperm cells and fertilization. A (too) high level of uterine activity probably reduces uptake of semen by the uterine horns and increases the risk of reflux. An important issue in the concept of sperm transport is the direction in which uterine contractions are propagated along the uterine horns. Waves of uterine contraction are thought to originate predominantly at the tubal and cervical ends of the uterine horns, these being the sites with the most pacemaker activity, at least during parturition [4]. Tubocervical directed contractions are thought to be important to expel seminal plasma after mating [38], but are probably also important in the distribution of semen over the two horns. After unilateral insemination, fertilization in the contralateral horn is similar to the fertilization rate in the ipsilateral horn, even with low sperm numbers (5 107) and a small volume (10 ml) [37]. Recently, however, evidence has also been provided for transport of sperm through the abdominal cavity to the contralateral oviduct (E. Martinez, personal communications). Cervico-tubal contractions are thought to be important in the transport of semen to the sperm reservoirs. Propagation of contractions depends on communication between myometrial cells, which is increased during estrus, when the number of gap junctions is increased due to the high level of estrogens [10,11]. We hypothesise that observations on the direction of uterine contractions probably depend on the site of measurement. In the tubal end of the horn, for example, cervically directed contractions probably dominate, because this site is located nearest to the tubal pacemaker zone. Contractions originating cervically are less likely to be propagated all the way to this site than contractions originating tubally. Studies on the direction of uterine contractions report tubo-cervical, cervico-tubal, and undirected contractions [2,3,5]. Another factor complicating studies on the direction of uterine contractions is the interpretation of pressure recordings. When several contractions follow closely in time, it is difficult to identify in what direction the contractions pass the recording sensors Nevertheless, reports on the direction of contractility claim that the direction of contractions is a coordinated process, which depends on the stage of estrus [2,3,5] and can be affected by stimuli involved in mating [3]. In summary, there is evidence for tubo-cervical, cervico-tubal, and undirected contractility, which are observed simultaneously throughout estrus. More information is needed to elucidate the coordination of contractility during different stages of estrus and the effects of external stimuli.
5. Conclusions Spontaneous uterine activity increases during estrus and has a function in the transport of sperm cells to the oviducts. This is particularly important when other factors around insemination, like semen dose, time of insemination relative to ovulation, etc., limit the number of sperm cells that survive until the moment of fertilization. The variation in uterine activity between sows is consistent throughout estrus and may contribute to the variation in fertilization results between sows. Stimulation of uterine contractility around insemination can improve sperm transport to the oviducts, but can also affect sperm
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transport and fertilization negatively. The latter occurs when contractility is stimulated to such an extent that the uptake of semen by the genital tract is obstructed and reflux of semen is increased. Introduction of a boar during insemination is probably an adequate way of stimulating contractility because oxytocin release is effectively induced in all sows and because boar presence selectively stimulates contractility in those sows with low spontaneous uterine activity. Future research should focus on the direction of uterine contractions and its regulation, and on the importance of separate components of the complex of boar stimuli for uterine contractility.
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