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Boar sperm with defective motility are discriminated in the backflow moments after insemination
Theriogenology 83 (2015) 655–661
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Boar sperm with defective motility are discriminated in the backflow moments after insemination I. Hernández-Caravaca a, b, C. Soriano-Úbeda a, c, C. Matás a, c, M.J. Izquierdo-Rico c, d, F.A. García-Vázquez a, c, * a
Department of Physiology, Faculty of Veterinary Science, International Excellence Campus for Higher Education and Research “Campus Mare Nostrum”, University of Murcia, Murcia, Spain Boehringer-Ingelheim S.A., Barcelona, Spain c Institute for Biomedical Research of Murcia (IMIB-Arrixaca), Murcia, Spain d Department of Cell Biology and Histology, Faculty of Medicine, University of Murcia, Murcia, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2014 Received in revised form 31 October 2014 Accepted 31 October 2014
During insemination, a large number of spermatozoa are deposited in the female genital tract, but a very low percentage is able to colonize the site of fertilization. The influx of neutrophils into the uterine lumen and semen reflux (backflow) are known mechanisms that decrease the number of spermatozoa within the uterus. No report has attempted to ascertain whether the backflow is a random or selective process of the spermatozoa. In this work, sows were inseminated using two populations of spermatozoa in the same proportion: (1) unstained spermatozoa with high motility and (2) stained spermatozoa with low, medium, or high motility. Volume, number, and percentage of stained spermatozoa were evaluated in the backflow (collected at 0–15, 16–30, and 31–60 minutes after insemination). This article provides evidence that (1) the motility characteristics of the spermatozoa do not influence the percentage of sows with backflow, the volume and number of spermatozoa in the backflow; (2) the discarding of spermatozoa in the backflow is not specific during the first moments after insemination (0–15 minutes), whereas later (16–60 minutes), spermatozoa with defective motility (low and medium groups) are discarded in a higher proportion than high group in the backflow ([16–30 minutes: low, 85.13 4.32%; medium, 72.99 5.05%; and high, 54.91 2.38%; P < 0.0001; 31–60 minutes: low, 87.16 6.01%; medium, 87.02 4.01%; and high, 59.35 2.86%; P ¼ 0.001]). Spermatozoa with poor motility are discarded in the backflow probably as a selective process, on the part of the female genital tract or as a result of the intrinsic low spermatozoa motility. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Backflow Motility Spermatozoa Porcine Uterus selection
1. Introduction When the spermatozoa are deposited in the female tract either as a result of natural or artificial insemination (AI), they have to travel a long way until they reach the ampullar-isthmic junction [1]. The process of sperm
* Corresponding author. Tel.: þ34 868 888009; fax: þ34 868 884147. E-mail address: [email protected] (F.A. García-Vázquez). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.10.032
transport through the uterus and oviduct is complex and involves dynamic interactions between spermatozoa and the female genital tract. Sperm transport to the site of fertilization is thought to be a combination of both passive and active transport. The spermatozoa encounter different environments within the female reproductive tract. The initial steps of sperm transport mediated by the female tract (passive transport) seem to be most important in the transport from the site of deposition to the proximal uterus and the uterotubal junction (UTJ) [2]. The passive part of
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sperm transport is probably because of the flow of fluid (i.e., extender, seminal plasma, or uterine fluid) and contractile movement of the uterine horns during the estrous period [3]. Active sperm transport resulting from the intrinsic movement of sperm cells seems to be important for their migration from the proximal uterus to the UTJ and the oviduct [4,5], although uterine contractions may also be involved [3]. Several studies have indicated that to reach the oviductal environment, spermatozoa have to be intact and endowed with appropriate motility [6–9]. Whatever the case, sperm transport is rapid, and within minutes of insemination, spermatozoa are found in the oviducts [1,10]. During porcine insemination, a large number of sperm are deposited in the female tract but only a few 1000 reach the oviduct. For e.g., if a sow is cervical inseminated with 3000 106 spermatozoa (100% of deposited spermatozoa), about 142 103 will reach the UTJ and 1.5 103 the sperm reservoir (SR) in the caudal part of the isthmus [11], which represents 0.0047% and 0.00005%, respectively, of the total spermatozoa deposited. The fact that most of the spermatozoa are lost during their passage through the female tract suggests that the uterus has certain mechanisms to discard and/or select spermatozoa. Two of the mechanisms known to be involved in spermatozoa losses are the influx of leukocytes from blood to the lumen of the uterus and backflow. Leukocytes are mainly polymorphonuclear neutrophils (PMNs) and the result of their influx into the uterine lumen is that the spermatozoa are phagocytized [12–14]. It has been hypothesized that PMNs take part in sperm cell selection, removing superfluous, nonmotile, or damaged spermatozoa [15]. Whether sperm cell phagocytosis is a selective or random process is still open to question [12]. Another known mechanism that provokes a reduction in sperm population in the female tract is backflow, which considerably reduces the volume and number of spermatozoa within 60 minutes of cervical AI (CAI; 54% and 25%, respectively) and post-cervical AI (post-CAI; 39% and 15%, respectively) [16]. Although there is some evidence that the spermatozoa in the backflow have been submitted to a selective process [16], further studies need to be performed to clarify how the spermatozoa are selected in the uterus on their way to the oviduct. The presence of different subpopulations in mammalian ejaculate (reviewed by Holt and Van Look [17]) has a functional implication although the exact role is still unknown. There is a lack of evidence concerning the effect that nonfunctional sperm (in terms of motility) will have on their selection in the uterus immediately after insemination. The hypothesis is that nonmotile or low-motile sperm are selected after sperm deposition and then discarded through the backflow. This hypothesis is based on our previous finding that spermatozoa of lower quality were found in the backflow in comparison with those in the original dose [16]. The present study evaluates the influence of different levels of sperm motility in the insemination doses on the volume, number, and type of spermatozoa (based on their motility characteristics) collected in the backflow at different times. The porcine industry is developing new biotechnologies such as sex-sorting sperm and freezing
thawing or sperm-mediated gene transfer that require significantly reduced sperm doses for AI. For these new procedures to be successful, new approaches, such as the use of post-CAI, are necessary [16,18–22] as this will decrease the number of spermatozoa used per dose. Besides, the quality of sperm obtained by sex sorting or freezing thawing is compromised [23], so it is important to know how the uterus selects sperm when the number of quality cells deposited is reduced. 2. Materials and methods 2.1. Ethics statement The experimental procedures for the use of animals were approved by the Ethical Committee of the University of Murcia. 2.2. Experimental design Nine ejaculates from seven boars (Duroc) of proven fertility and 45 multiparous sows (Landrace Large White) were used in this study. The females were inseminated using a total of 1500 106 spermatozoa in 25 mL. Every insemination sperm dose was composed of two parts from the same ejaculate: (1) 750 106 unstained sperm in 12.5 mL with high motility (>70% motility) and (2) 750 106 spermatozoa in 12.5 mL previously stained with Hoechst and with different levels of motility (low, medium, or high). The average of the different sperm motility populations used for this study was as follows: low, 7.50 4.33%; medium, 42.50 12.50%; and high, 75.00 2.88% of motility (%). The spermatozoa were stained to clearly and objectively identify the sperm population collected in the backflow because their motility characteristics can change between insemination and collection. Two different populations were used: unstained sperm of high motility mixed just before post-CAI with stained sperm of low, medium, or high motility. An aliquot of 500 mL of the dose after mixing and before insemination was kept to count the percentage of sperm stained (approximately 50%) as a control. The backflow was collected in each individual sow at different times (0–15, 16–30, and 31–60 minutes) from the beginning of insemination. Once in the laboratory (within 1 hour of collecting the backflow), the volume (% of the dosage), concentration of spermatozoa (% of the dosage), and number of stained sperm (%) collected in the backflow were evaluated. 2.3. Sperm collection Semen was obtained from each boar once a week using the gloved-hand technique and filtered to remove the gel. The number of spermatozoa in the ejaculates was counted in a hemocytometer (Neubauer counting chamber; VWR International, Haasrode, Belgium). Only ejaculates with a rich fraction volume, 75 mL or greater; concentration, 200 106 spermatozoa/mL or greater; motility, 70% or greater; and total abnormalities, 20% or lesser were used in this study. Immediately after evaluation, and before
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transport to the laboratory, the semen was diluted at 38 C by adding 100 mL of commercial extender (Zoosperm ND4, Import-Vet S.A., Barcelona, Spain). 2.4. Semen processing On reaching the laboratory, the semen doses were adjusted. First, the number of spermatozoa was calculated by a SpermaCue photometer (Minitüb, Germany), and later, a commercial extender was added (when necessary) until the concentration required (100 106 spermatozoa/mL) was reached. Tubes of extended semen (9900 mL) were supplemented (or not, see Section 2.2) with 100 mL of 5 mg/ mL Hoechst 33342 (Sigma–Aldrich, Madrid, Spain) and kept in the dark and at room temperature for 15 minutes (modified from Bathgate et al. [24]). The sperm samples (unstained and stained sperm) were carefully centrifuged twice (600 g for 5 minutes) and readjusted to 50 106 spermatozoa/mL. These semen samples were stored at 16 C until they were used (within 2 hours after processing). 2.5. Analysis of seminal motion parameters The percentage (%) of motile sperm was determined by placing two aliquots on warm glass slides (38 C) which were examined immediately at 100 magnification under a microscope coupled to a thermal plate (Eclipse E200; Nikon, Tokyo, Japan). The analyses were run by the same person to avoid intertester variability. 2.6. Insemination procedure Multiparous sows (average parity number: 6.00 1.6 [mean standard deviation]) used for breeding were weaned 28 days after farrowing. Estrus detection was performed twice daily by experienced workers by allowing sows nose-to-nose contact with mature boars and applying back pressure. The occurrence of estrus was defined by the standing reflex in front of a teaser boar and reddening and swelling of the vulva. Only sows with clear signs of estrus were used for the experiment. The AI procedure was performed as described by Hernández-Caravaca et al. [16]. The sow perineal area was carefully cleaned with 0.5% chlorhexidine gluconate wipes (Despro, Import-Vet S.A.). Post-CAI was achieved with a combined catheter-cannula kit (Soft & Quick; Import-Vet S.A., Barcelona, Spain), which consists of a 72-cm flexible cannula inserted into a conventional cervical catheter. Sows were inseminated 24 hours after estrus detection with doses of 1.5 109 spermatozoa in 25 mL. The sperm dose was introduced quickly (taking only a few seconds). 2.7. Backflow collection and analyses After insemination, the perineal area was thoroughly cleaned and dried. The semen backflow was collected in human colostomy bags (fixed around the vulva and secured with tape) [16,25] during the 60 minutes after insemination. If a sow urinated into the colostomy bag or the colostomy bag was damaged, the corresponding value was deleted from the data.
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2.7.1. Backflow volume and sperm concentration assay The colostomy bag was emptied into a graduated tube to measure the volume, whereas the sperm concentrations were assessed in triplicate using a Neubauer counting chamber. 2.7.2. Analysis of stained sperm The semen collected in the backflow was fixed in 0.3% formaldehyde saline solution, mounted on glass slides, and examined under an epifluorescence microscope with phase contrast at 1000 magnification. A minimum of 200 spermatozoa per slide were analyzed and categorized as stained and unstained according to the sperm UV fluorescence. 2.8. Statistical analysis All statistical analyses were performed using SPSS v.15 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean standard error of the mean and were analyzed by one-way ANOVA. When ANOVA revealed a significant effect, values were compared using the least significant difference pairwise multiple comparison post hoc test (Tukey). Differences were considered statistically significant at P < 0.05. 3. Results There was no difference in the parity number of the sows assigned to each group (low, 5.75 2.06; medium, 6.17 1.47; and high, 6.00 2.00). From the total number of sows used in this experiment (N ¼ 45), five (11.36%) urinated at some stage during the collection and the bags were discarded for the calculation of backflow data. So, finally 39 multiparous sows were used as follows: 13 for the lowmotility group, 14 for medium, and 12 for the high group. When the sows were inseminated with the different experimental doses (low, medium, and high-level motility; see Section 2.2.), no differences were found in the percentage of sows showing backflow (Fig. 1) at the different times analyzed. At 0 to 15 minutes after insemination, the percentage of sows with backflow ranged from 44% (low group) to 75% (high group). At 16 to 30 minutes, the percentage of sows with backflow ranged from 55% (low group) to 100% (medium group) and at 31 to 60 minutes, the percentage ranged from 88.89% (low group) to 100% (medium and high groups; Fig. 1). When the volume was analyzed, no statistical differences were found between the three experimental groups (range, 52.62%–58.17%; Fig. 2A). Similarly, no differences were observed when the three experimental groups were compared in the collection periods (0–15 minutes: range, 6.80%–13.86%; 16–30 minutes: range, 15.25%–25.30%; and 31–60 minutes: range, 17.45%–26.30%; Fig. 2B). As regards the spermatozoa, 10.73% to 24.24% of the number of inseminated spermatozoa was recovered in the backflow within 60 minutes of insemination. No differences were observed between the three experimental groups analyzed (Fig. 2C). When the spermatozoa in the backflow were related with the collection time (Fig. 2D), the percentage of spermatozoa was similar in the three experimental groups with no significant difference between any of the periods studied. Regardless of the experimental group
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spermatozoa, the results were similar for all groups (data not shown), indicating that the dye did not interfere. In addition, the percentage of stained sperm in the dose after mixing was evaluated before AI to test the relative proportions of sperm. The data indicated similar sperm population ratios (51.10 0.77% of the spermatozoa was stained in a total of 6600 cells analyzed; control group in Fig. 3A). When the percentage of stained sperm in the backflow was calculated based on the total amount of spermatozoa found in the backflow after 60 minutes, the data indentified statistical differences (Fig. 3A). The high group showed the lowest percentage of spermatozoa stained compared with the medium and low groups (P < 0.0001) but similar to the control. The differences were significant between the low and medium groups (P ¼ 0.009). The data were also analyzed for each of the backflow collection times (Fig. 3B). During the first 15 minutes after AI, the percentage of sperm stained in the backflow was similar in all three experimental groups. However, the percentage of sperm stained collected in the second period (16–30 minutes) differed among groups. The highest rate was observed in the low group with significant differences from the medium (P ¼ 0.014) and high (P < 0.0001) groups. Finally, the percentage of stained sperm found in the last period measured (31–60 minutes) reached the highest level in the low and medium groups, compared with the high group (P ¼ 0.001; Fig. 3B, C). 4. Discussion
Fig. 1. Sows with backflow (%) in the experimental groups (low, medium, and high) at different times.
and collection time, the percentage of spermatozoa recovered in the backflow was always less than 9% (Fig. 2D). However, sperm motility in the insemination dose was seen to influence the type of spermatozoa (percentage of stained sperm) collected in the backflow. When the motility of high-motility subpopulations (unstained vs. stained) was compared after centrifugation and processing to test whether the dye used (Hoechst 33342) affected the
In mammals, fertilization is the natural transplantation of cells from males to females and in general terms, involves the deposition of a high number of spermatozoa in the female reproductive tract. During their journey, the spermatozoa are exposed to different environments (uterine and oviductal fluid), types of cell (PMNs, uterine and oviductal epithelial cells), and actions (such as uterine contractions or fluid flow). As a consequence, only very few spermatozoa reach the SR in the oviduct, suggesting that spermatozoa are selected in the female genital tract. Some of the sperm are eliminated by the PMNs present in the uterine lumen and some are refluxed (backflow) after insemination (reviewed by Soriano-Úbeda et al. [5]). However, it is not clear whether the reduction in the sperm population within the uterus is selective and depends on sperm characteristics or is a random process. In the present study, the influence of sperm motility (percentage of motile sperm) on the volume and number of spermatozoa and type of spermatozoa (stained or unstained, based on their motility characteristics) in the backflow was evaluated. For this purpose, three experimental groups with two different subpopulations (unstained sperm of high motility and stained sperm of low, medium, or high motility) were used for insemination. The results showed that the sperm motility level does not influence the volume or number of spermatozoa in the backflow; however, a higher proportion of poorly motile sperm than of spermatozoa of adequate motility is refluxed 15 minutes after insemination. The backflow of semen is a physiological process in pigs because it happens after both natural [26] and artificial
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Fig. 2. The volume and number of spermatozoa in the backflow after insemination. Backflow volume measured 60 minutes after the beginning of insemination (A) and at different times (B) in the three experimental groups (low, medium, and high). The data are expressed as a percentage of volume used in the insemination dose (mean standard error of the mean [SEM]). Spermatozoa in the backflow measured 60 minutes after the beginning of insemination (C) and at different times (D) in the three experimental groups (low, medium, and high). The data are expressed as a percentage of spermatozoa used in the insemination dose (mean SEM). In figure labels (B and D), the low group is represented by a dark gray bar, medium group by a white bar, and high group by a light gray bar.
[16,21,25] insemination. In the present study, spermatozoa were deposited directly into the uterus to avoid the cervix barrier, as has been previously reported (reviewed by Holt [27]). Moreover, uterine deposition permits better and faster distribution of the spermatozoa in the female genital tract and also prevents excessive semen backflow during the insemination process [11]. As mentioned in other reports [16,21,25], most of the sows showed backflow at some time after insemination. The number of spermatozoa, volume of seminal plasma, and/or extender in the case of AI have been reported to influence the backflow [16,21,28] probably because of an increase in uterine contractility. In addition, in this experiment, there was no correlation between the motility of the spermatozoa and the number of sows with backflow. In the present study, greater than 50% of the volume and 15% to 20% of the spermatozoa deposited were lost within 1 hour of insemination. Sperm motility did not influence the volume or number of spermatozoa collected in the backflow after 60 minutes or during the different periods studied (Fig. 2). Some studies have attempted to evaluate how the presence of altered spermatozoa might modify the uterus in some aspects [29–33] although there are no data on how sperm quality could influence the backflow characteristics. Some of the above reports have evaluated endometrium changes in several species after insemination with dead spermatozoa [31–33] but further studies are necessary to clarify the findings. For the study, cells were stained and submitted to a double centrifugation. The effect of exposure of boar sperm
to Hoechst 33342 has been studied previously [34], and no differences were found in the motility patterns after exposure to a similar concentration as that used in the present study. Even fertility parameters were similar among sows inseminated with sperm stained with Hoechst 33342 and those inseminated with unstained sperm [34]. Under normal circumstances, a low number of spermatozoa are sufficient for fertilization, and these establish themselves in the oviduct during the first hour after insemination [1]. In the caudal part of the isthmus, spermatozoa bind to epithelial cells and can be stored until just before ovulation with no reduction in their fertilizing ability until ovulation. For this reason, this part of the oviduct is named the SR [35]. The rapid removal of spermatozoa is thought to prevent the acquisition of immune responses against sperm [36]. According to the results of the present study, during the first 15 minutes after insemination (rapid removal of spermatozoa), the spermatozoa are “unselected” with the same proportion of sperm (unstained spermatozoa with high motility and stained spermatozoa with low, medium or high motility) being found in the backflow (Fig. 3). One interpretation of this result is that during the first 15 minutes after sperm deposition in the female, uterus contractions transport the spermatozoa in two directions: upward (toward the oviduct) and downward (toward the vagina), and this mechanism could provoke the backflow of “unselected” sperm. But, after this time, the more capable spermatozoa reach the oviduct and establish an SR by attaching
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Fig. 3. Stained sperm in the backflow measured 60 minutes after the beginning of insemination (A) and at different times (B) in the three experimental groups (low, medium, and high). The data are expressed as a percentage of stained sperm (mean SEM). In figure label B, low group is represented by dark gray bar, medium group by white bar, and high group by light gray bar. The control bar in figure label A represents the percentage of stained sperm after mixing the doses (unstained and stained sperm) before insemination. a,b,cIndicate differences (P < 0.05) among experimental groups. (C) Spermatozoa progressing (toward oviduct direction) and returning (toward uterus body) in the female reproductive tract after insemination. The left part of the scheme represents the first 15 minutes after deposition when the spermatozoa are refluxed (in the backflow) regardless of their motility. The right part depicts sperm selection after at least 16 minutes in the female genital tract, in which the spermatozoa are selected on the basis of their motion characteristics, while those with defective motility reflux.
themselves to the oviductal epithelial cells, whereas other viable sperm populations are directly attached to the uterine epithelial cells. In both cases, the spermatozoa with normal motility probably because attached to the uterus, while contractions continue in the female tract, as a result of which unattached spermatozoa are expelled through the backflow, i.e., less motile and less able to fertilize sperm. It was previously reported that more defective spermatozoa are found in the backflow within 1 hour of insemination than are deposited, suggesting preselection of the sperm in the uterus [16] or that the spermatozoa with altered quality (i.e., low motility or abnormalities) are refluxed because of their incapacity to advance within the uterus. It has been suggested that viable sperm attach themselves to uterine epithelial cells as part of the spermatozoa selection process in the female genital tract [13], in a mechanism that has been proposed as a selective negative process, preventing part of the sperm population reaching the oviduct [13]. The binding of spermatozoa to the epithelial cells could induce signals that favor subsequent inflammatory responses [13]. This is supported by the observations of Rozeboom et al. [37], who describe how the influx of neutrophils increased after insemination with spermatozoa in extender compared with extender without spermatozoa. In addition, spermatozoa with good motility
may act to keep sperm in suspension in fluids of the female tract, thereby reducing adhesion to the endometrium (reviewed by Hunter [4]). The distribution of dead and live spermatozoa within the female tract has been evaluated previously. After insemination of gilts with 50% of dead sperm and 50% of live sperm, the number of live sperm collected in the uterus and oviduct increased with the time after insemination [29]. In the first 15 minutes after insemination, the proportions of dead and live sperm collected were similar (around 50% of each), whereas from 15 minutes to 60 minutes, the number of live sperm collected in the uterus increased (58%–77%). These results agree with the present data, probably as a result of the removal of dead sperm from the female tract through the backflow or due to the loss of active transport of dead spermatozoa, among other possible reasons such as in the uterine fluid [38]. Another option is that the UTJ blocks the transit of spermatozoa with a defective flagellar function, as was shown in mouse [6,9] and that these spermatozoa are cleaned from the uterus tract by the mechanisms that have already been mentioned. The mechanisms involved in sperm selection are complex, and probably many factors are involved, such as physical size, shape, longevity within the female reproductive tract, and metabolic efficiency, as well as the ability to interact with the
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female reproductive tract and overcome the obstacles that the spermatozoa encounter on their way to fertilize the oocyte [39]. 4.1. Conclusions
[14]
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[16]
Increasing our knowledge of backflow and how spermatozoa are selected in the uterus should help improve AI efficiency. The initial hypothesis is confirmed because the spermatozoa with adequate motility remained within the uterus, ensuring fertilization. This study provides evidence of how sperm motility influences their selection within the uterus after insemination. A high proportion of low-motility sperm are found in the backflow after 15 minutes of insemination, before which time, the spermatozoa collected in the backflow are unspecific and independent of motility.
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Acknowledgments
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This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER; grant number, AGL2012-40180-C03-01-02). Author contributions: conceived and designed the experiments: F.A. García-Vázquez and C. Matás; performed the experiments: F.A. García-Vázquez, C. Matás, C. SorianoÚbeda, I. Hernández-Caravaca, and M.J. Izquierdo-Rico; analyzed the data: F.A. García-Vázquez; and wrote the paper: F.A. García-Vázquez, C. Matás, I. Hernández-Caravaca, and M.J. Izquierdo-Rico. References
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Boar sperm with defective motility are discriminated in the backflow moments after insemination I. Hernández-Caravaca a, b, C. Soriano-Úbeda a, c, C. Matás a, c, M.J. Izquierdo-Rico c, d, F.A. García-Vázquez a, c, * a
Department of Physiology, Faculty of Veterinary Science, International Excellence Campus for Higher Education and Research “Campus Mare Nostrum”, University of Murcia, Murcia, Spain Boehringer-Ingelheim S.A., Barcelona, Spain c Institute for Biomedical Research of Murcia (IMIB-Arrixaca), Murcia, Spain d Department of Cell Biology and Histology, Faculty of Medicine, University of Murcia, Murcia, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2014 Received in revised form 31 October 2014 Accepted 31 October 2014
During insemination, a large number of spermatozoa are deposited in the female genital tract, but a very low percentage is able to colonize the site of fertilization. The influx of neutrophils into the uterine lumen and semen reflux (backflow) are known mechanisms that decrease the number of spermatozoa within the uterus. No report has attempted to ascertain whether the backflow is a random or selective process of the spermatozoa. In this work, sows were inseminated using two populations of spermatozoa in the same proportion: (1) unstained spermatozoa with high motility and (2) stained spermatozoa with low, medium, or high motility. Volume, number, and percentage of stained spermatozoa were evaluated in the backflow (collected at 0–15, 16–30, and 31–60 minutes after insemination). This article provides evidence that (1) the motility characteristics of the spermatozoa do not influence the percentage of sows with backflow, the volume and number of spermatozoa in the backflow; (2) the discarding of spermatozoa in the backflow is not specific during the first moments after insemination (0–15 minutes), whereas later (16–60 minutes), spermatozoa with defective motility (low and medium groups) are discarded in a higher proportion than high group in the backflow ([16–30 minutes: low, 85.13 4.32%; medium, 72.99 5.05%; and high, 54.91 2.38%; P < 0.0001; 31–60 minutes: low, 87.16 6.01%; medium, 87.02 4.01%; and high, 59.35 2.86%; P ¼ 0.001]). Spermatozoa with poor motility are discarded in the backflow probably as a selective process, on the part of the female genital tract or as a result of the intrinsic low spermatozoa motility. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Backflow Motility Spermatozoa Porcine Uterus selection
1. Introduction When the spermatozoa are deposited in the female tract either as a result of natural or artificial insemination (AI), they have to travel a long way until they reach the ampullar-isthmic junction [1]. The process of sperm
* Corresponding author. Tel.: þ34 868 888009; fax: þ34 868 884147. E-mail address: [email protected] (F.A. García-Vázquez). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.10.032
transport through the uterus and oviduct is complex and involves dynamic interactions between spermatozoa and the female genital tract. Sperm transport to the site of fertilization is thought to be a combination of both passive and active transport. The spermatozoa encounter different environments within the female reproductive tract. The initial steps of sperm transport mediated by the female tract (passive transport) seem to be most important in the transport from the site of deposition to the proximal uterus and the uterotubal junction (UTJ) [2]. The passive part of
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sperm transport is probably because of the flow of fluid (i.e., extender, seminal plasma, or uterine fluid) and contractile movement of the uterine horns during the estrous period [3]. Active sperm transport resulting from the intrinsic movement of sperm cells seems to be important for their migration from the proximal uterus to the UTJ and the oviduct [4,5], although uterine contractions may also be involved [3]. Several studies have indicated that to reach the oviductal environment, spermatozoa have to be intact and endowed with appropriate motility [6–9]. Whatever the case, sperm transport is rapid, and within minutes of insemination, spermatozoa are found in the oviducts [1,10]. During porcine insemination, a large number of sperm are deposited in the female tract but only a few 1000 reach the oviduct. For e.g., if a sow is cervical inseminated with 3000 106 spermatozoa (100% of deposited spermatozoa), about 142 103 will reach the UTJ and 1.5 103 the sperm reservoir (SR) in the caudal part of the isthmus [11], which represents 0.0047% and 0.00005%, respectively, of the total spermatozoa deposited. The fact that most of the spermatozoa are lost during their passage through the female tract suggests that the uterus has certain mechanisms to discard and/or select spermatozoa. Two of the mechanisms known to be involved in spermatozoa losses are the influx of leukocytes from blood to the lumen of the uterus and backflow. Leukocytes are mainly polymorphonuclear neutrophils (PMNs) and the result of their influx into the uterine lumen is that the spermatozoa are phagocytized [12–14]. It has been hypothesized that PMNs take part in sperm cell selection, removing superfluous, nonmotile, or damaged spermatozoa [15]. Whether sperm cell phagocytosis is a selective or random process is still open to question [12]. Another known mechanism that provokes a reduction in sperm population in the female tract is backflow, which considerably reduces the volume and number of spermatozoa within 60 minutes of cervical AI (CAI; 54% and 25%, respectively) and post-cervical AI (post-CAI; 39% and 15%, respectively) [16]. Although there is some evidence that the spermatozoa in the backflow have been submitted to a selective process [16], further studies need to be performed to clarify how the spermatozoa are selected in the uterus on their way to the oviduct. The presence of different subpopulations in mammalian ejaculate (reviewed by Holt and Van Look [17]) has a functional implication although the exact role is still unknown. There is a lack of evidence concerning the effect that nonfunctional sperm (in terms of motility) will have on their selection in the uterus immediately after insemination. The hypothesis is that nonmotile or low-motile sperm are selected after sperm deposition and then discarded through the backflow. This hypothesis is based on our previous finding that spermatozoa of lower quality were found in the backflow in comparison with those in the original dose [16]. The present study evaluates the influence of different levels of sperm motility in the insemination doses on the volume, number, and type of spermatozoa (based on their motility characteristics) collected in the backflow at different times. The porcine industry is developing new biotechnologies such as sex-sorting sperm and freezing
thawing or sperm-mediated gene transfer that require significantly reduced sperm doses for AI. For these new procedures to be successful, new approaches, such as the use of post-CAI, are necessary [16,18–22] as this will decrease the number of spermatozoa used per dose. Besides, the quality of sperm obtained by sex sorting or freezing thawing is compromised [23], so it is important to know how the uterus selects sperm when the number of quality cells deposited is reduced. 2. Materials and methods 2.1. Ethics statement The experimental procedures for the use of animals were approved by the Ethical Committee of the University of Murcia. 2.2. Experimental design Nine ejaculates from seven boars (Duroc) of proven fertility and 45 multiparous sows (Landrace Large White) were used in this study. The females were inseminated using a total of 1500 106 spermatozoa in 25 mL. Every insemination sperm dose was composed of two parts from the same ejaculate: (1) 750 106 unstained sperm in 12.5 mL with high motility (>70% motility) and (2) 750 106 spermatozoa in 12.5 mL previously stained with Hoechst and with different levels of motility (low, medium, or high). The average of the different sperm motility populations used for this study was as follows: low, 7.50 4.33%; medium, 42.50 12.50%; and high, 75.00 2.88% of motility (%). The spermatozoa were stained to clearly and objectively identify the sperm population collected in the backflow because their motility characteristics can change between insemination and collection. Two different populations were used: unstained sperm of high motility mixed just before post-CAI with stained sperm of low, medium, or high motility. An aliquot of 500 mL of the dose after mixing and before insemination was kept to count the percentage of sperm stained (approximately 50%) as a control. The backflow was collected in each individual sow at different times (0–15, 16–30, and 31–60 minutes) from the beginning of insemination. Once in the laboratory (within 1 hour of collecting the backflow), the volume (% of the dosage), concentration of spermatozoa (% of the dosage), and number of stained sperm (%) collected in the backflow were evaluated. 2.3. Sperm collection Semen was obtained from each boar once a week using the gloved-hand technique and filtered to remove the gel. The number of spermatozoa in the ejaculates was counted in a hemocytometer (Neubauer counting chamber; VWR International, Haasrode, Belgium). Only ejaculates with a rich fraction volume, 75 mL or greater; concentration, 200 106 spermatozoa/mL or greater; motility, 70% or greater; and total abnormalities, 20% or lesser were used in this study. Immediately after evaluation, and before
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transport to the laboratory, the semen was diluted at 38 C by adding 100 mL of commercial extender (Zoosperm ND4, Import-Vet S.A., Barcelona, Spain). 2.4. Semen processing On reaching the laboratory, the semen doses were adjusted. First, the number of spermatozoa was calculated by a SpermaCue photometer (Minitüb, Germany), and later, a commercial extender was added (when necessary) until the concentration required (100 106 spermatozoa/mL) was reached. Tubes of extended semen (9900 mL) were supplemented (or not, see Section 2.2) with 100 mL of 5 mg/ mL Hoechst 33342 (Sigma–Aldrich, Madrid, Spain) and kept in the dark and at room temperature for 15 minutes (modified from Bathgate et al. [24]). The sperm samples (unstained and stained sperm) were carefully centrifuged twice (600 g for 5 minutes) and readjusted to 50 106 spermatozoa/mL. These semen samples were stored at 16 C until they were used (within 2 hours after processing). 2.5. Analysis of seminal motion parameters The percentage (%) of motile sperm was determined by placing two aliquots on warm glass slides (38 C) which were examined immediately at 100 magnification under a microscope coupled to a thermal plate (Eclipse E200; Nikon, Tokyo, Japan). The analyses were run by the same person to avoid intertester variability. 2.6. Insemination procedure Multiparous sows (average parity number: 6.00 1.6 [mean standard deviation]) used for breeding were weaned 28 days after farrowing. Estrus detection was performed twice daily by experienced workers by allowing sows nose-to-nose contact with mature boars and applying back pressure. The occurrence of estrus was defined by the standing reflex in front of a teaser boar and reddening and swelling of the vulva. Only sows with clear signs of estrus were used for the experiment. The AI procedure was performed as described by Hernández-Caravaca et al. [16]. The sow perineal area was carefully cleaned with 0.5% chlorhexidine gluconate wipes (Despro, Import-Vet S.A.). Post-CAI was achieved with a combined catheter-cannula kit (Soft & Quick; Import-Vet S.A., Barcelona, Spain), which consists of a 72-cm flexible cannula inserted into a conventional cervical catheter. Sows were inseminated 24 hours after estrus detection with doses of 1.5 109 spermatozoa in 25 mL. The sperm dose was introduced quickly (taking only a few seconds). 2.7. Backflow collection and analyses After insemination, the perineal area was thoroughly cleaned and dried. The semen backflow was collected in human colostomy bags (fixed around the vulva and secured with tape) [16,25] during the 60 minutes after insemination. If a sow urinated into the colostomy bag or the colostomy bag was damaged, the corresponding value was deleted from the data.
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2.7.1. Backflow volume and sperm concentration assay The colostomy bag was emptied into a graduated tube to measure the volume, whereas the sperm concentrations were assessed in triplicate using a Neubauer counting chamber. 2.7.2. Analysis of stained sperm The semen collected in the backflow was fixed in 0.3% formaldehyde saline solution, mounted on glass slides, and examined under an epifluorescence microscope with phase contrast at 1000 magnification. A minimum of 200 spermatozoa per slide were analyzed and categorized as stained and unstained according to the sperm UV fluorescence. 2.8. Statistical analysis All statistical analyses were performed using SPSS v.15 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean standard error of the mean and were analyzed by one-way ANOVA. When ANOVA revealed a significant effect, values were compared using the least significant difference pairwise multiple comparison post hoc test (Tukey). Differences were considered statistically significant at P < 0.05. 3. Results There was no difference in the parity number of the sows assigned to each group (low, 5.75 2.06; medium, 6.17 1.47; and high, 6.00 2.00). From the total number of sows used in this experiment (N ¼ 45), five (11.36%) urinated at some stage during the collection and the bags were discarded for the calculation of backflow data. So, finally 39 multiparous sows were used as follows: 13 for the lowmotility group, 14 for medium, and 12 for the high group. When the sows were inseminated with the different experimental doses (low, medium, and high-level motility; see Section 2.2.), no differences were found in the percentage of sows showing backflow (Fig. 1) at the different times analyzed. At 0 to 15 minutes after insemination, the percentage of sows with backflow ranged from 44% (low group) to 75% (high group). At 16 to 30 minutes, the percentage of sows with backflow ranged from 55% (low group) to 100% (medium group) and at 31 to 60 minutes, the percentage ranged from 88.89% (low group) to 100% (medium and high groups; Fig. 1). When the volume was analyzed, no statistical differences were found between the three experimental groups (range, 52.62%–58.17%; Fig. 2A). Similarly, no differences were observed when the three experimental groups were compared in the collection periods (0–15 minutes: range, 6.80%–13.86%; 16–30 minutes: range, 15.25%–25.30%; and 31–60 minutes: range, 17.45%–26.30%; Fig. 2B). As regards the spermatozoa, 10.73% to 24.24% of the number of inseminated spermatozoa was recovered in the backflow within 60 minutes of insemination. No differences were observed between the three experimental groups analyzed (Fig. 2C). When the spermatozoa in the backflow were related with the collection time (Fig. 2D), the percentage of spermatozoa was similar in the three experimental groups with no significant difference between any of the periods studied. Regardless of the experimental group
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spermatozoa, the results were similar for all groups (data not shown), indicating that the dye did not interfere. In addition, the percentage of stained sperm in the dose after mixing was evaluated before AI to test the relative proportions of sperm. The data indicated similar sperm population ratios (51.10 0.77% of the spermatozoa was stained in a total of 6600 cells analyzed; control group in Fig. 3A). When the percentage of stained sperm in the backflow was calculated based on the total amount of spermatozoa found in the backflow after 60 minutes, the data indentified statistical differences (Fig. 3A). The high group showed the lowest percentage of spermatozoa stained compared with the medium and low groups (P < 0.0001) but similar to the control. The differences were significant between the low and medium groups (P ¼ 0.009). The data were also analyzed for each of the backflow collection times (Fig. 3B). During the first 15 minutes after AI, the percentage of sperm stained in the backflow was similar in all three experimental groups. However, the percentage of sperm stained collected in the second period (16–30 minutes) differed among groups. The highest rate was observed in the low group with significant differences from the medium (P ¼ 0.014) and high (P < 0.0001) groups. Finally, the percentage of stained sperm found in the last period measured (31–60 minutes) reached the highest level in the low and medium groups, compared with the high group (P ¼ 0.001; Fig. 3B, C). 4. Discussion
Fig. 1. Sows with backflow (%) in the experimental groups (low, medium, and high) at different times.
and collection time, the percentage of spermatozoa recovered in the backflow was always less than 9% (Fig. 2D). However, sperm motility in the insemination dose was seen to influence the type of spermatozoa (percentage of stained sperm) collected in the backflow. When the motility of high-motility subpopulations (unstained vs. stained) was compared after centrifugation and processing to test whether the dye used (Hoechst 33342) affected the
In mammals, fertilization is the natural transplantation of cells from males to females and in general terms, involves the deposition of a high number of spermatozoa in the female reproductive tract. During their journey, the spermatozoa are exposed to different environments (uterine and oviductal fluid), types of cell (PMNs, uterine and oviductal epithelial cells), and actions (such as uterine contractions or fluid flow). As a consequence, only very few spermatozoa reach the SR in the oviduct, suggesting that spermatozoa are selected in the female genital tract. Some of the sperm are eliminated by the PMNs present in the uterine lumen and some are refluxed (backflow) after insemination (reviewed by Soriano-Úbeda et al. [5]). However, it is not clear whether the reduction in the sperm population within the uterus is selective and depends on sperm characteristics or is a random process. In the present study, the influence of sperm motility (percentage of motile sperm) on the volume and number of spermatozoa and type of spermatozoa (stained or unstained, based on their motility characteristics) in the backflow was evaluated. For this purpose, three experimental groups with two different subpopulations (unstained sperm of high motility and stained sperm of low, medium, or high motility) were used for insemination. The results showed that the sperm motility level does not influence the volume or number of spermatozoa in the backflow; however, a higher proportion of poorly motile sperm than of spermatozoa of adequate motility is refluxed 15 minutes after insemination. The backflow of semen is a physiological process in pigs because it happens after both natural [26] and artificial
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Fig. 2. The volume and number of spermatozoa in the backflow after insemination. Backflow volume measured 60 minutes after the beginning of insemination (A) and at different times (B) in the three experimental groups (low, medium, and high). The data are expressed as a percentage of volume used in the insemination dose (mean standard error of the mean [SEM]). Spermatozoa in the backflow measured 60 minutes after the beginning of insemination (C) and at different times (D) in the three experimental groups (low, medium, and high). The data are expressed as a percentage of spermatozoa used in the insemination dose (mean SEM). In figure labels (B and D), the low group is represented by a dark gray bar, medium group by a white bar, and high group by a light gray bar.
[16,21,25] insemination. In the present study, spermatozoa were deposited directly into the uterus to avoid the cervix barrier, as has been previously reported (reviewed by Holt [27]). Moreover, uterine deposition permits better and faster distribution of the spermatozoa in the female genital tract and also prevents excessive semen backflow during the insemination process [11]. As mentioned in other reports [16,21,25], most of the sows showed backflow at some time after insemination. The number of spermatozoa, volume of seminal plasma, and/or extender in the case of AI have been reported to influence the backflow [16,21,28] probably because of an increase in uterine contractility. In addition, in this experiment, there was no correlation between the motility of the spermatozoa and the number of sows with backflow. In the present study, greater than 50% of the volume and 15% to 20% of the spermatozoa deposited were lost within 1 hour of insemination. Sperm motility did not influence the volume or number of spermatozoa collected in the backflow after 60 minutes or during the different periods studied (Fig. 2). Some studies have attempted to evaluate how the presence of altered spermatozoa might modify the uterus in some aspects [29–33] although there are no data on how sperm quality could influence the backflow characteristics. Some of the above reports have evaluated endometrium changes in several species after insemination with dead spermatozoa [31–33] but further studies are necessary to clarify the findings. For the study, cells were stained and submitted to a double centrifugation. The effect of exposure of boar sperm
to Hoechst 33342 has been studied previously [34], and no differences were found in the motility patterns after exposure to a similar concentration as that used in the present study. Even fertility parameters were similar among sows inseminated with sperm stained with Hoechst 33342 and those inseminated with unstained sperm [34]. Under normal circumstances, a low number of spermatozoa are sufficient for fertilization, and these establish themselves in the oviduct during the first hour after insemination [1]. In the caudal part of the isthmus, spermatozoa bind to epithelial cells and can be stored until just before ovulation with no reduction in their fertilizing ability until ovulation. For this reason, this part of the oviduct is named the SR [35]. The rapid removal of spermatozoa is thought to prevent the acquisition of immune responses against sperm [36]. According to the results of the present study, during the first 15 minutes after insemination (rapid removal of spermatozoa), the spermatozoa are “unselected” with the same proportion of sperm (unstained spermatozoa with high motility and stained spermatozoa with low, medium or high motility) being found in the backflow (Fig. 3). One interpretation of this result is that during the first 15 minutes after sperm deposition in the female, uterus contractions transport the spermatozoa in two directions: upward (toward the oviduct) and downward (toward the vagina), and this mechanism could provoke the backflow of “unselected” sperm. But, after this time, the more capable spermatozoa reach the oviduct and establish an SR by attaching
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Fig. 3. Stained sperm in the backflow measured 60 minutes after the beginning of insemination (A) and at different times (B) in the three experimental groups (low, medium, and high). The data are expressed as a percentage of stained sperm (mean SEM). In figure label B, low group is represented by dark gray bar, medium group by white bar, and high group by light gray bar. The control bar in figure label A represents the percentage of stained sperm after mixing the doses (unstained and stained sperm) before insemination. a,b,cIndicate differences (P < 0.05) among experimental groups. (C) Spermatozoa progressing (toward oviduct direction) and returning (toward uterus body) in the female reproductive tract after insemination. The left part of the scheme represents the first 15 minutes after deposition when the spermatozoa are refluxed (in the backflow) regardless of their motility. The right part depicts sperm selection after at least 16 minutes in the female genital tract, in which the spermatozoa are selected on the basis of their motion characteristics, while those with defective motility reflux.
themselves to the oviductal epithelial cells, whereas other viable sperm populations are directly attached to the uterine epithelial cells. In both cases, the spermatozoa with normal motility probably because attached to the uterus, while contractions continue in the female tract, as a result of which unattached spermatozoa are expelled through the backflow, i.e., less motile and less able to fertilize sperm. It was previously reported that more defective spermatozoa are found in the backflow within 1 hour of insemination than are deposited, suggesting preselection of the sperm in the uterus [16] or that the spermatozoa with altered quality (i.e., low motility or abnormalities) are refluxed because of their incapacity to advance within the uterus. It has been suggested that viable sperm attach themselves to uterine epithelial cells as part of the spermatozoa selection process in the female genital tract [13], in a mechanism that has been proposed as a selective negative process, preventing part of the sperm population reaching the oviduct [13]. The binding of spermatozoa to the epithelial cells could induce signals that favor subsequent inflammatory responses [13]. This is supported by the observations of Rozeboom et al. [37], who describe how the influx of neutrophils increased after insemination with spermatozoa in extender compared with extender without spermatozoa. In addition, spermatozoa with good motility
may act to keep sperm in suspension in fluids of the female tract, thereby reducing adhesion to the endometrium (reviewed by Hunter [4]). The distribution of dead and live spermatozoa within the female tract has been evaluated previously. After insemination of gilts with 50% of dead sperm and 50% of live sperm, the number of live sperm collected in the uterus and oviduct increased with the time after insemination [29]. In the first 15 minutes after insemination, the proportions of dead and live sperm collected were similar (around 50% of each), whereas from 15 minutes to 60 minutes, the number of live sperm collected in the uterus increased (58%–77%). These results agree with the present data, probably as a result of the removal of dead sperm from the female tract through the backflow or due to the loss of active transport of dead spermatozoa, among other possible reasons such as in the uterine fluid [38]. Another option is that the UTJ blocks the transit of spermatozoa with a defective flagellar function, as was shown in mouse [6,9] and that these spermatozoa are cleaned from the uterus tract by the mechanisms that have already been mentioned. The mechanisms involved in sperm selection are complex, and probably many factors are involved, such as physical size, shape, longevity within the female reproductive tract, and metabolic efficiency, as well as the ability to interact with the
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female reproductive tract and overcome the obstacles that the spermatozoa encounter on their way to fertilize the oocyte [39]. 4.1. Conclusions
[14]
[15]
[16]
Increasing our knowledge of backflow and how spermatozoa are selected in the uterus should help improve AI efficiency. The initial hypothesis is confirmed because the spermatozoa with adequate motility remained within the uterus, ensuring fertilization. This study provides evidence of how sperm motility influences their selection within the uterus after insemination. A high proportion of low-motility sperm are found in the backflow after 15 minutes of insemination, before which time, the spermatozoa collected in the backflow are unspecific and independent of motility.
[20]
Acknowledgments
[21]
This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER; grant number, AGL2012-40180-C03-01-02). Author contributions: conceived and designed the experiments: F.A. García-Vázquez and C. Matás; performed the experiments: F.A. García-Vázquez, C. Matás, C. SorianoÚbeda, I. Hernández-Caravaca, and M.J. Izquierdo-Rico; analyzed the data: F.A. García-Vázquez; and wrote the paper: F.A. García-Vázquez, C. Matás, I. Hernández-Caravaca, and M.J. Izquierdo-Rico. References
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