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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Little is known about the response of the bitch's reproductive tract to mating or of the role of male accessory gland secretions in the female. In this clinical study, the component stimuli causing mating-induced uterine contractions were investigated in 64 bitches. Basal uterine contractions were present during oestrus and a significant increase in the frequency of contractions was observed during natural mating. Neither teasing with a male nor stimulation of the vagina or cervix by vaginal or transcervical insemination (TCI) caused an increase in the frequency of uterine contractions. Increased contractions were however present after both vaginal and transcervical insemination when the vestibule was distended, and dorsal wall of the vaginal was manually stimulated. Interestingly, this increase in uterine contractions was partially ameliorated when prostatic fluid was used as a flushing component following transcervical insemination. Two further studies performed with 72 bitches of which 18 were each inseminated transcervically with fresh or frozen semen flushed into the uterus with either saline or prostatic fluid demonstrated that prostatic fluid significantly increased the pregnancy rate and litter size of both groups. There are important mechanisms regulating the transport and elimination of sperm from the bitch's reproductive tract. Whilst physical aspects of coitus are undoubtedly involved in initiating uterine contractions, prostatic fluid appears to have an important role in modulating uterine contractions and fertility.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

In the bitch, uterine contractions occur during oestrus and are specifically increased during mating, although the initiating factors are unknown (England et al. 2006, 2012a). Uterine contractions presumably aid in sperm transport and are part of the mechanism by which sperm are rapidly propelled to the tips of the uterine horns (Evans 1933) where they establish an intimate attachment with uterine epithelium to form a sperm reservoir (Burgess 2000). Sperm ‘bound’ to oestrus uterine epithelium have increased longevity (Pacey et al. 2000), which is important for the extended fertilization period in this species (England et al. 2006). Uterine contractions in the bitch are also important for elimination of excess and dead spermatozoa, seminal fluid, bacteria and other contaminants that enter the uterus at mating; the frequency of uterine contractions has been related to the rate of fluid clearance from the uterus (England et al. 2012a).

Entrance of sperm into the bitch's uterus results in a transient increase in uterine artery blood velocity and a reduction in the resistance index, changes which indicate vasodilation (England et al. 2012b). This response is associated with an influx of polymorphonuclear neutrophils (PMNs), and although the inflammatory response is important for removing uterine contaminants, PMNs reduce the ability of sperm to attach to uterine epithelium (England et al. 2012b) and as such could have a deleterious effect upon fertility. Fortunately, the impact of PMNs reducing sperm-epithelial attachment is ameliorated by seminal plasma and prostatic fluid (PF), both of which appear to reduce the attachment of PMNs to sperm (England et al. 2012b).

Whilst the need for an inflammatory response to eliminate uterine contaminants is clear, the contradictory dilemma is the need to ensure that inflammation does not negatively impact sperm transport nor reduce the population of fertile sperm. The principal methods for sperm transport and elimination are both increased uterine contractions (Portus et al. 2005), but at least for the bitch, there are no studies that define the mechanisms of induction of mating-induced uterine contractions, how these might be modulated nor the effect that such modulation might have upon fertility. The purpose of this study was therefore to investigate the stimuli that initiate uterine contractions at coitus, with particular reference to the role of PF, and furthermore to investigate the impact of PF on pregnancy rate and litter size.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Ethical approval was granted by the School of Veterinary Medicine and Science, University of Nottingham. All bitches were presented for mating or artificial insemination (AI) and were clinically normal and had a normal endometrial appearance following detailed transabdominal ultrasound examination of the uterine body and distal uterine horns using a 10 MHz transducer (Pie Veterinary Ltd, Crawley, West Sussex). All Labrador and Golden retrievers were subject to the same feeding and management regimes. Uterine contractions were measured on the first day of mating/insemination designated as when plasma progesterone concentrations first exceeded 10 ng/ml using an ELISA (Premate, Synbiotics). Mating or insemination was repeated the following day using the same technique but no further data were collected. Pregnancy rate and litter size data were provided by the owner.

All male dogs used for breeding or fresh semen AI had achieved a pregnancy in the previous 2 months and had normal semen quality according to established criteria (England 1999). Semen collection for AI was by masturbation in the presence of a teaser bitch. The three fractions were collected separately using glass funnels, and the first fraction was discarded.

For frozen semen inseminations, PF was collected from six additional normal clinical cases and was pooled, divided into 1.0 ml aliquants and frozen at −20 °C until required. Fertility of dogs providing frozen semen was not always known, and semen quality post-thaw was variable; however, the number of straws used was adjusted so that approximately 150 × 106 sperm with normal progressive motility were inseminated resulting in a volume between 2.5 and 4.0 ml. All semen samples and fluids were held at 39°C until immediately prior to use.

Study 1. Stimuli that initiate mating-induced uterine contractions

The uterine body of 64 normal bitches (aged 25–40 months) that were divided into eight groups each containing 4 Labrador retrievers, 2 golden retrievers, 1 bullmastiff and 1 greyhound was examined in the longitudinal plane using a 10 MHz transducer 30 min before and immediately after mating or insemination stimuli. The 64 bitches were mated or inseminated with semen from 22 males of which six were common to each group (2 Labrador retrievers, 2 golden retrievers, 1 bullmastiff and 1 greyhound). Semen used for insemination was evaluated according to the method of England (1999), and the number of morphologically normal motile sperm was recorded prior to use. For dogs naturally mated, semen had been collected 5–7 days before the mating and semen evaluation performed at that time.

The frequency of uterine contractions was counted in a 20 min time period (England et al. 2006). Each group received a different stimulus designated as (i) Teasing with a male: a male was allowed to tease and mount the female for 10 min without intromission; uterine contractions were measured immediately after cessation of teasing (these bitches were mated after the ultrasound examination but no further measurements were made; seven of the eight bitches become pregnant), (ii) Natural mating: bitches were mated and uterine contractions were measured 2 min after the start of the copulatory ‘tie’, (iii) Vaginal AI: an approximate 2 min endoscopic examination of the vagina was undertaken followed by placement of a vaginal insemination catheter (Osiris, IMV) with inflation of the cuff in the cranial mid vagina and infusion of the second fraction of the ejaculate; uterine contractions were measured immediately after infusion with the catheter left in place, (iv) Vaginal AI with vaginal stimulation: endoscopy, placement of the Osiris catheter and infusion was as previously described but in addition the vagina was stimulated by placing two lubricated gloved fingers through the vestibule and into the vagina to allow ‘stroking’ of the dorsal vaginal wall at a rate of 20 stimuli per minute for 20 min (appropriate stimuli was confirmed by vestibular and perineal contractions of the bitch); uterine contractions were measured immediately after infusion and throughout the vaginal stimulation (v) TCI with saline flush: a six French gauge catheter was placed through the cervix using a rigid cystourethroscope with a 6 mm sheath (Wolf) and the second fraction was inseminated and flushed through the catheter using 1.0 ml phosphate-buffered saline (PBS); uterine contractions were measured immediately after the procedure was completed, (f) TCI with PF flush: TCI was performed and semen was flushed into the uterus with 1.0 ml homologous PF at 39°C; uterine contractions were measured immediately after the procedure was completed, (vi) TCI with PBS flush and vaginal stimulation: insemination and PBS flushing was performed and was followed immediately by vaginal stimulation as previously described; uterine contractions were measured 2 min after stimulation commenced, (vii) TCI with PF flush and vaginal stimulation: insemination, flushing with homologous PF and stimulation were performed as previously described; uterine contractions were measured 2 min after stimulation commenced.

Study 2. Effect of prostatic fluid on pregnancy rate and litter size following fresh semen AI

To evaluate the effect of PF on fertility, 36 clinical cases (aged 25–42 months) presented for AI with fresh semen were divided into two groups each containing 18 bitches as follows (i) Fresh semen TCI with PBS flush (9 Labrador retrievers, 3 golden retrievers, 4 bullmastiff and 2 greyhounds): the second fraction was inseminated transcervically and was flushed into the uterus using 1.0 ml PBS as previously described, (ii) Fresh semen TCI with PF flush (8 Labrador retrievers, 4 golden retrievers, 3 bullmastiff and 3 greyhounds): the second fraction was inseminated and was flushed using 1.0 ml homologous PF. The 36 bitches were inseminated with semen from 22 different males of which 14 were common to the two groups (6 Labrador retrievers, 3 golden retrievers, 2 bullmastiff and 2 greyhounds). Semen was evaluated according to the method of England (1999), and the number of morphologically normal motile sperm was recorded prior to insemination.

All samples were held at 39°C until insemination, which involved manual stimulation of the vagina and inseminations were performed on two consecutive days as previously described.

Study 3. Effect of prostatic fluid on pregnancy rate and litter size following frozen semen AI

To evaluate the effect of PF on fertility, 36 clinical cases (aged 28–45 months) presented for AI with frozen semen were divided into two groups each containing 18 bitches as follows: (i) Frozen semen TCI with PBS flush (4 Labrador retrievers, 4 golden retrievers, 4 bullmastiff, 2 greyhounds and 4 beagle): approximately 140 × 106 progressively motile sperm was inseminated and was flushed using 1.0 ml PBS, (ii) Frozen semen TCI with PF flush (4 Labrador retrievers, 3 golden retrievers, 4 bullmastiff, 2 greyhounds and 5 beagles). The 36 bitches were inseminated with semen from 19 different males of which 17 were common to the two groups (4 Labrador retrievers, 3 golden retrievers, 4 bullmastiff, 2 greyhounds and 4 beagles). Approximately 140 × 106 progressively motile morphologically normal sperm was inseminated and flushed using 1.0 ml heterologous frozen-thawed PF. All samples were held at 39°C until insemination, which involved manual stimulation of the vagina, and inseminations were performed on two consecutive days as previously described.

Statistical evaluation

Means (±SE) and percentages were calculated for each group, and data were analysed using analysis of variance for uterine contraction and semen quality data, and Fishers exact and chi-square test for fertility data, using InStat3 (GraphPad, La Jolla, CA, USA). Values were considered significant when p < 0.05.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Study 1. Stimuli that initiate mating-induced uterine contractions

Each bitch had an apparently normal oestrus and was mated/inseminated as planned. There was no significant difference in mean age between the eight groups.

There was no statistical difference in the mean number of progressively motile and morphologically normal sperm for any of the seven groups; mean numbers were 387 ± 98 × 106, 421 ± 154 × 106, 338 ± 182 × 106, 299 ± 111 × 106, 302 ± 89 × 106, 372 ± 143 × 106 and 331 ± 134 × 106, for natural mating (data from 5 to 7 days before the mating), Vaginal AI, Vaginal AI with vaginal stimulation, TCI with PBS flush, TCI with PF flush, TCI with PBS flush and vaginal stimulation, and TCI with PF flush and vaginal stimulation, respectively.

Basal uterine contractions were noted in all bitches and there were no significant differences in the mean frequency of contractions between the groups. Uterine contractions were more difficult to detect when there were vestibular/perineal contractions, but careful observation allowed differentiation of the latter, which caused movement of all tissues rather than just the uterus. The mean frequency of basal contractions for the 64 bitches was 6.62 ± 0.18 (SE) contractions/20 min. Teasing did not increase the frequency of contractions (mean, 6.62 ± 0.32 contractions/20 min), but there was a significant increase during mating (mean 12.62 ± 0.38 contractions/20 min). Vaginal insemination did not cause a significant increase in the frequency of contractions above baseline (mean, 8.12 ± 0.23 contractions/20 min) except when there was concomitant additional manual stimulation of the vagina (mean, 11.12 ± 0.22 contractions/20 min). Similarly, TCI did not cause a change in the frequency of uterine contractions when either PBS or PF flush was used (mean 7.87 ± 0.22 and 7.37 ± 0.18 contractions/20 min for PBS and PF, respectively) except when PBS flush was used combined with manual vaginal stimulation (mean, 11.25 ± 0.38 contractions/20 min). Interestingly, the increase in uterine contractions caused by TCI and manual vaginal stimulation was partially ameliorated when PF flush was used rather than PBS flush (mean, 9.25 ± 0.45 contractions/20 min; Fig. 1).

image

Figure 1. The mean frequency of uterine contractions recorded with ultrasound from groups of eight bitches after different components of the mating and artificial insemination process. AI, artificial insemination; TCI, transcervical insemination; PBS, phosphate-buffered saline; PF, prostatic fluid

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Pregnancy rate and litter size were not significantly different across the seven groups, although values were numerically smaller for vaginal insemination without vaginal stimulation. Pregnancy rate and mean litter size were 87.5% and 6.7 ± 0.8 pups for natural mating, 62.5% and 5.6 ± 0.5 pups for vaginal insemination, 75.0% and 6.2 ± 0.4 pups for vaginal insemination with vaginal stimulation, 100% and 6.9 ± 0.6 pups for TCI with PBS flush, 100% and 7.3 ± 0.3 pups for TCI with PF flush, 100% and 7.1 ± 0.4 pups for TCI with PBS flush and vaginal stimulation and 100% and 7.9 ± 0.3 pups for TCI with PF flush and vaginal stimulation.

Study 2. Effect of prostatic fluid on pregnancy rate and litter size following fresh semen AI

Each bitch had an apparently normal oestrus and was inseminated as planned. There was no significant difference in mean age between the two groups.

Quality of semen used for insemination of the two groups was not statistically different; a mean of 355 ± 121 × 106 progressively motile and morphologically normal sperm were inseminated into bitches flushed with homologous PF, and a mean of 412 ± 102 × 106 progressively motile and morphologically normal sperm were inseminated into bitches flushed PBS.

Pregnancy rate was significantly higher for bitches inseminated with fresh semen with homologous PF flush (94.4%) compared with bitches inseminated with fresh semen with PBS flush (61.1%; Fig. 2). Litter size was significantly greater for bitches inseminated with fresh semen with homologous PF flush (7.47 ± 0.19 pups) compared with bitches inseminated with fresh semen with PBS flush (6.27 ± 0.30 pups; Fig. 2).

image

Figure 2. The percentage pregnancy rate and mean litter size from groups of 18 bitches inseminated transcervically with fresh semen followed by flushing with either phosphate-buffered saline (PBS) or prostatic fluid (PF) and manual stimulation of the vagina

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Study 3. Effect of prostatic fluid on pregnancy rate and litter size following frozen semen AI

For bitches inseminated with frozen-thawed semen, pregnancy rate was significantly higher for bitches inseminated with heterologous PF flush (83.3%) compared with bitches inseminated with PBS flush (44.4%; Fig. 3). Similarly, for bitches inseminated with frozen-thawed semen, pregnancy rate was significantly higher for bitches inseminated with heterologous PF flush (5.13 ± 0.24 pups) compared with bitches inseminated with PBS flush (3.62 ± 0.45 pups; Fig. 3).

image

Figure 3. The percentage pregnancy rate and mean litter size from groups of 18 bitches inseminated transcervically with frozen-thawed semen followed by flushing with either phosphate-buffered saline (PBS) or prostatic fluid (PF) and manual stimulation of the vagina

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Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Spontaneous uterine contractions during oestrus are common in many species, although they are not detected in all individuals (Langendijk et al. 2002). Mating-induced contractions may be caused by endometrial prostaglandins (induced by components of seminal plasma), tactile stimulation and oxytocin release in the female (Claus 1990; Campbell and England 2004; Langendijk et al. 2005). Attention has been given to the role of uterine contractions for elimination of uterine contaminants (Nikolakopoulos and Watson 1999); indeed, this has also been purported in the bitch (England et al. 2012a), but there has been surprisingly little investigation of the role of uterine contractions in sperm transport.

Mating-induced uterine contractions have been measured in both mares and bitches (Campbell and England 2002; England et al. 2006). In mares, amplitude, frequency and duration of contractions have been recorded, but in bitches, measurement has only been made of frequency. In the present study, the mean frequency of basal and mating-induced uterine contractions was similar to previous reports (Wheaton et al. 1988; England et al. 2006). We did not measure the direction of contractions but in several species mating produces both cervicotubal and tubocervical contractions (Crane and Martin 1991; Campbell and England 2006) enabling distribution of sperm to the oviduct (Katila et al. 2000). A further limitation of the present study was that some measurements were made during stimulation (e.g. during mating), whilst others were made after the period of stimulation ceased (e.g. after TCI). Nevertheless, the data clearly demonstrate a significant mating-induced increase in uterine contractions that were at least partially mimicked by manual stimulation of the vagina. Neither teasing with a male nor vaginal or transcervical insemination without manual stimulation produced a significant increase in contractions.

Interesting in the present study was the smaller increase in vaginal-stimulated uterine contractions when TCI was followed by PF rather than PBS flush. It is commonly proposed that male accessory gland secretions increase uterine contraction (Crane and Martin 1991), although our findings are similar to those of Portus et al. (2005) who showed that uterine contractions were decreased in mares inseminated with seminal plasma. The present study used PF rather than seminal plasma, and clearly, there is a large component of PF in equine seminal plasma; further work is needed to elucidate possible differential actions of various accessory gland secretions in different species. Interestingly, fluid from the different accessory glands of mice and rats have contrasting effects on fertility (Pang et al. 1979; Queen et al. 1981).

The role of PF in regulating the response of the female tract to mating is intriguing as are the findings of increased pregnancy rate and litter size following PF flushing of fresh and frozen-thawed semen. Dog PF is known to have a deleterious effect on sperm after prolonged incubation (England and Allen 1992), but has previously been shown to enhance fertility following insemination of frozen-thawed semen (Nöthling and Volkmann 1993; Nöthling et al. 2005). Similar observations have been made about seminal plasma when inseminating mares and ewes (Alghamdi et al. 2004; Lopez-Perez and Perez-Clariget 2012). We have previously demonstrated that PF increases the attachment of sperm to uterine epithelium likely by two mechanisms one of which involves the reduction of sperm attachment to PMNs (England et al. 2012b). The present study demonstrates a clear and unambiguous improvement in fertility when PF was used to flush semen into the uterus, and we hypothesize that this is in part associated with reduced uterine contractions in the immediate period after mating as well as the aforementioned effects on sperm attachment to uterine epithelium.

The present study offers interesting insights to the mechanisms responsible for initiating mating-induced uterine contractions in the bitch and documents a pivotal role for prostatic fluid in regulating both these events and fertility.

Conflicts of interest

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

None of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of this work.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
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