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Biology of Reproduction 67, 1073-1079 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Sperm Binding to Epithelial Oviduct Explants in Bulls with Different Nonreturn Rates Investigated with a New In Vitro Model1

Ingrid M.C. De Pauwa, Ann Van Soom2,a, Hans Laevensa, Steven Verberckmoesa, and Aart de Kruifa

a Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke 9820, Belgium


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of ;lt20 000 µm2 provided more consistent results than did explants with a surface area of >100 000 µm2. A positive association was found between the loge transformed number of spermatozoa bound to 0.1 mm2 oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

female reproductive tract, fertilization, oviduct, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most mammals, ejaculated spermatozoa pass through the female reproductive tract to form a functional sperm reservoir in the lower segment of the oviduct [1]. In cattle, it takes about 6–8 h before enough spermatozoa have reached this sperm reservoir to ensure fertilization [2]. Spermatozoa attach to the apical plasma membrane of the ciliated and secretory epithelial cells [3]. This attachment is mediated by fucose recognition [4]. Other factors that could be involved in the establishment of a sperm reservoir are impedance of sperm movement by oviductal mucus [5] and low patency of the oviduct due to edematous mucosa and tightly contracted myosalpinx [6].

The reservoir acts to ensure that enough fertile spermatozoa are available in the oviduct when ovulation occurs. Bovine spermatozoa can remain arrested in the isthmus for >=18 h and only detach from the epithelium near the time of ovulation [7]. This prolonged sperm survival is especially important in species with longer estrous periods, such as the horse [8], but might also be of interest for cattle, which are inseminated early in estrus. If a bull is unable to populate the sperm reservoir with a sufficient number of spermatozoa or for a sufficient period of time, fertility could be adversely affected. The field fertility of a given bull is expressed by the nonreturn rate (NRR), which is defined as the proportion of cows that were inseminated and did not return for another service within 56 days [9]. Differences probably exist among bulls in their capacity to establish a sperm reservoir after mating or insemination, but it is difficult to detect these differences after matings and collection of oviducts because the number of sperm cells reported to reach the oviduct in vivo has differed considerably among studies and within experiments [5, 10, 11].

To determine whether the capacity to establish a reservoir is indicative of fertility, we searched for an in vitro approach to studying the capacity of sperm to bind to oviductal epithelium. In vitro sperm membrane integrity can be extended by coincubating the spermatozoa with oviductal epithelium [12]. To investigate this sperm binding in more detail, we needed a reliable method to determine the number of sperm cells attached to oviductal epithelium. The aim of this study was to set up a model to quantify sperm binding to oviductal epithelium by combining fluorescent staining of sperm and image analysis. After standardizing, this model was tested to evaluate whether sperm binding density could be used to predict in vivo bull fertility. For this purpose, sperm binding density to oviductal explants was assessed in sires with known NRRs, which are indicative of in vivo fertility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media

Chemicals and media were obtained from Sigma Chemical Company (Bornem, Belgium) and Gibco Invitrogen Corporation (Merelbeke, Belgium). The following media were used in this study. Hepes-buffered Tyrode albumin lactate pyruvate (TALP) medium contained 114 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 2.1 mM CaCl2, 0.4 mM MgCl2, 2 mM NaHCO3, 0.2 mM sodium pyruvate, 10 mM sodium lactate, 10 µg/ml gentamycin sulfate, 10 mM Hepes, and 3 mg/ml BSA. In vitro fertilization (IVF) medium contained TALP without Hepes supplemented with 25 mM NaHCO3, 0.2 mM sodium pyruvate, 10 mM sodium lactate, 10 µg/ml gentamycin sulfate, and 6 mg/ml fatty acid-free BSA. Heparin was omitted because it would prevent capacitation and heparin-induced sperm release from oviduct explants [13, 14]. A modified bicarbonate buffered tissue culture medium (TCM-199) was supplemented with 0.2 mM sodium pyruvate, 0.4 mM glutamine, 50 µg/ml gentamycin sulfate, and 2.5 µg/ml fungizone. Fetal calf serum (FCS; 10%) was added as noted.

Media were passed through sterile 0.22-µm Acrodisc Syringe low-protein binding filters (Millipore Corp., New Bedford, MA) before being used.

Preparation of Spermatozoa

For standardization of the model to study sperm binding to oviductal epithelium in vitro, fresh and frozen-thawed semen of a 2-yr-old Red Pied bull were used.

To study the relationship between NRR and sperm binding to oviductal epithelium, we used frozen-thawed semen from 10 Holstein Friesian bulls with known fertility (expressed as 56-day NRR) varying from 52.8% to 69.9%. The difference between the highest and the lowest NRR was 17%. This was the maximum range available from the Artificial Insemination (AI) center. The NRRs were based on a total of 1884 first inseminations with frozen-thawed semen with a minimum of 163 AIs per bull. Straws were generously supplied by VRV (Flemish Cattle Breeding Association, Belgium). Artificial inseminations were performed by experienced veterinarians in dairy herds during fall and winter of 2000–2001. Uncorrected average NRRs were used, but bias was kept to a minimum by using sires with comparable numbers of services, which were used in the winter period (less use of natural breeding). Semen was processed for freezing as described by Den Daas et al. [9]. Frozen-thawed semen obtained from the same ejaculate was used both for the investigation of sperm binding to oviductal epithelium as for the determination of the NRR. Two straws of frozen semen were thawed in a water bath at 37°C for at least 30 sec and washed twice in 5 ml of Hepes-TALP solution by centrifugation at 720 x g for 10 min at room temperature. After removing the supernatant, the concentration of the spermatozoa was measured with a Bürker chamber. The final sperm concentration added to the oviduct explants was 106 spermatozoa/ml. This concentration was similar to the concentration used in related studies [15, 16]. The progressive motility was subjectively assessed by visual estimation under a light microscope (Leica DMR, Van Hopplynus N.V., Brussels, Belgium; 200x) equipped with a stage warmer (37°C). The nucleic stains SYBR-14 and propidium iodide (PI) (LIVE/DEAD Sperm Viability Kit; Molecular Probes, Leiden, The Netherlands) were used for analyzing the membrane integrity of the spermatozoa just before coincubation with the oviduct explants.

Collection and Processing of Oviducts for Culture

Bovine epithelium explants were collected and prepared according to a modification of the procedure of Madison et al. [17]. Oviducts were transported to the laboratory in physiological saline (0.9% NaCl) on ice. Upon arrival, they were dissected free of the surrounding tissues and rinsed in Hepes-TALP. The dissected oviduct was cut open longitudinally with sterile scissors under a laminar flow hood and was lightly scraped with a sterile scalpel to collect epithelial tissue. Sheets of epithelial cells were transferred to a 15-ml tube containing 5 ml Hepes-TALP. After initial sedimentation, the supernatant was removed, and 5 ml of fresh Hepes-TALP was added to the pellet. The same volume was removed again after the second sedimentation. The last washing was performed in modified TCM-199 medium + 10% FCS. The supernatant was removed, and the cell sheets were disaggregated into smaller pieces by passing once through a 21-gauge needle attached to a 1-ml syringe.

Two hundred microliters of sedimented oviduct explants was added to 5 ml modified TCM-199 medium + 10% FCS and cultured overnight in a 50-ml flask. After overnight culture, the oviduct cells had formed clumps or worms with beating cilia. The medium containing the oviduct explants was transferred to a 15-ml tube. After sedimentation, the supernatant was removed, and 50 µl of the oviduct explants was added to 250 µl of the prepared semen sample and 200 µl of medium in a four-well culture plate.

Quantification of Sperm Binding to Oviductal Epithelium

The number of spermatozoa bound to oviductal epithelium was determined using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1; Molecular Probes) to stain mitochondria. This fluorophore exhibits potential-dependent accumulation in mitochondria and can reversibly change its emission from green to red with increasing transmembrane electrical potential [18]. JC-1 was combined with the classical dead cell stain PI to identify membrane-damaged spermatozoa [19] (Fig. 1). Density of bound spermatozoa on one side of the oviduct explant was determined after 30 min, 24 h, and 48 h by means of fluorescence microscopy performed by using the Leica DMR microscope equipped with an excitation filter of 450–490 nm from a 100 W mercury lamp and examined at a magnification of 100x (explants with a surface area of >100 000 µm2) or 400x (explants with a surface area of <20 000 µm2). The surface of the oviduct explant was measured with the Image Database program (Leica, Van Hopplynus N.V.).



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  		FIG. 1. Fluorescence image of spermatozoa bound to oviduct explant stained with JC-1, which labels the mitochondria of spermatozoa yellow and that of oviduct cells green, and with PI, which stains the heads of membrane-damaged spermatozoa red. x400

Experimental Design

Standardization of a model to study sperm binding to oviduct epithelium Frozen-thawed and fresh spermatozoa from a Red Pied bull were washed and incubated separately with oviduct explants at a concentration of 106 spermatozoa/ml medium. Small and large oviduct explants were equally distributed between fresh and frozen-thawed spermatozoa.

The different media, Hepes-TALP, IVF-TALP, TCM-199 + 10% FCS, and TCM-199 were incubated at 38.5°C in 5% CO2 except for Hepes-TALP (38.5°C, in air). After 30 min, 24 h, and 48 h of coincubation, at least 10 oviduct explants were stained with JC-1 and PI for 15 min. The oviduct explants with bound spermatozoa were first transferred twice to fresh medium by means of a micropipette (Unopette Capillary Pipettes; Becton Dickinson, Franklin Lakes, NJ) to remove unbound spermatozoa and then placed on a glass slide and viewed under a Leica DMR fluorescence microscope (100x or 400x) equipped with a stage warmer (37°C). The surface area of 10 oviduct explants per point of time and per medium was measured, and the number of spermatozoa bound to one side of the oviduct explant was calculated. The size of the explants varied between 9200 µm2 and 137 219 µm2. The number of spermatozoa bound to 0.1 mm2 of explant surface was used as the parameter of binding capacity. The experiment was repeated three times. Each replicate was performed with oviduct explants from the same batch.

In a second experiment, the influence of the size of oviduct explants on sperm binding was examined. The binding of frozen-thawed spermatozoa to 10 oviduct explants with a surface area of <20 000 µm2 (Fig. 2A) and to 10 oviduct explants with a surface area of >100 000 µm2 (Fig. 2B) was compared after 30 min of coincubation. Sperm binding density was expressed as the number of spermatozoa bound to 0.1 mm2 of oviduct epithelium. The experiment was repeated three times. Each replicate was performed with oviduct explants from the same batch.



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  		FIG. 2. Bull spermatozoa are bound to oviduct explants and labeled with JC-1 and PI. A) Explant surface area of <20 000 µm2. x400. B) Explant surface of >100 000 µm2. x100

Relationship between NRR and capacity of sperm to bind to oviduct epithelial explants The optimized model was used to investigate whether there is a difference in capacity to bind to oviduct explants for frozen-thawed spermatozoa from 10 Holstein Friesian bulls with different NRRs (52.8–69.9%). The influence of the initial membrane integrity on the association between the loge transformed number of spermatozoa bound to 0.1 mm2 of oviduct epithelium and the NRR was also investigated. Membrane integrity of spermatozoa from each sperm sample was evaluated by fluorescence microscopy just before addition to the oviduct explants (LIVE/DEAD Sperm Viability Kit). This experiment was repeated three times.

Frozen-thawed spermatozoa were used for this experiment because all AIs were carried out with frozen-thawed spermatozoa. Only oviduct explants with a surface area of <20 000 µm2 (obtained by passing twice through a 26-gauge needle) were used. The mean values and ranges of size of oviduct explants were comparable for each bull.

Statistical Methods

The influence of different media and sperm preservation methods (fresh versus frozen thawed) on capacity of sperm to bind to epithelial oviduct explants was analyzed with the Mixed procedure of SAS version 8 (SAS Institute, Cary, NC). The outcome variable was expressed as the loge transformed number of spermatozoa bound per 0.1 mm2 of oviduct explant. Explanatory variables were the media and the sperm preservation method. Both explanatory variables were included as class variables. Time was considered a repeated measure and batch a random effect.

The influence of the size (small = <20 000 µm2; large = >100 000 µm2) of oviduct epithelial explants on sperm binding was analyzed by means of a two-sample t-test. Differences were considered significant at P = 0.05.

The influence of different bulls with given NNRs and membrane integrity rates on capacity of sperm to bind to epithelial oviduct explants was analyzed with the Mixed procedure of SAS version 8. The outcome variable was expressed as the loge transformed number of spermatozoa bound per 0.1 mm2 of oviduct explant. Explanatory variables were the membrane integrity rate (%) and the NRR (%). Both explanatory variables were included as continuous variables. Time was considered a repeated measure, and bull was a random effect. Regression lines of the number of spermatozoa bound to epithelial oviduct explants after 24 h against the NRR and for different membrane integrity rates (40%, 45%, ..., 85%) were calculated by using the ESTIMATE statement of the Mixed procedure.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
An additional experiment was conducted to investigate whether the number of oviduct explants present in a 50-µl sample could affect the number of sperm binding to each oviduct explant. The number of oviduct explants present in a 50-µl sample taken from a mixture of both large and small oviduct explants or from small oviduct explants only was counted. The results indicated that the mean number of oviduct explants was not significantly different between replicate samples (large and small oviduct explants: mean ± SEM = 182 ± 3, coefficient of variation = 0.06, n = 10; small oviduct explants: mean ± SEM = 247 ± 5, coefficient of variation = 0.06, n = 10).

Spermatozoa (106) were then coincubated with a culture of 200, 250, 275, and 300 small oviduct explants. The number of spermatozoa bound to 10 oviduct explants from each culture was counted after 30 min, 24 h, and 48 h. At each time point, no significant difference in the number of spermatozoa per 0.1 mm2 of explant was observed when the number of oviduct explants ranged between 200 and 300 (ANOVA, Scheffé test; data not shown).

Influence of Media and Sperm Preservation Method on Binding of Fresh and Frozen-Thawed Spermatozoa to Oviduct Explants

Significantly (P < 0.05; Table 1) more fresh spermatozoa were bound to oviduct explants incubated in Hepes-TALP or IVF-TALP than in the other media. Fresh spermatozoa remained bound to oviduct explants in Hepes-TALP in substantial numbers even after 48 h of incubation. For frozen-thawed spermatozoa, differences in sperm-oviduct binding were observed between Hepes-TALP and TCM-199 with or without addition of 10% FCS (P < 0.05; Table 1). The decrease in the number of frozen-thawed bound spermatozoa in Hepes-TALP differed (P < 0.05) from that in IVF-TALP over time. The binding capacity of fresh and frozen-thawed spermatozoa was not different (P = 0.12). Because more spermatozoa remained bound to oviduct explants incubated in Hepes-TALP after 48 h, this medium was used in the remaining experiments.


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  		TABLE 1. The effect of different media on sperm binding capacity after 30 min, 24 h, or 48 h of culture (geometric mean and 95% CI of the number of spermatozoa/0.1 mm2 of oviduct explant; three replicates, 10 observations/replicate). Sperm preservation method did not affect the overall or the time-dependent binding capacity within the same medium (P >= 0.05)

Influence of the Size of Oviduct Epithelial Explants on Sperm Binding

After 30 min of coincubation, significantly more spermatozoa (two-sample t-test, P < 0.001) were bound per 0.1 mm2 of oviduct epithelium when the surface area of the oviduct explants was <20 000 µm2 than when it was >100 000 µm2 (528 ± 70 and 85 ± 20, respectively). The coefficient of variation (CV) within the group of small oviduct explants (CV = 0.13) was smaller than that in the group of large oviduct explants (CV = 0.24). As a result, only oviduct explants with a surface area of <20 000 µm2 were used to investigate the relationship between NRR and sperm binding capacity.

Relationship Between NRR and Capacity of Sperm to Bind to Oviduct Epithelial Explants

The relation between the number of spermatozoa bound to 0.1 mm2 of oviductal epithelium and the NRR was dependent on time (Fig. 3). The number of bound spermatozoa decreased significantly over time (P < 0.001).



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  		FIG. 3. Binding capacity of spermatozoa of each bull to oviduct epithelium after 30 min, 24 h, and 48 h of culture (geometric mean of three replicates). Bulls are ranked according to increasing NRR. NRR and membrane integrity rates per bull are presented in Table 2

The association between sperm binding capacity and NRR was also dependent on membrane integrity of the spermatozoa. At the start of coincubation, the initial mean percentage of membrane-intact spermatozoa in the tested sperm samples measured with SYBR-14 and PI after thawing and washing of the spermatozoa ranged from 34% to 79% (Table 2).


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  		TABLE 2. Percentage of membrane-intact spermatozoa in the tested sperm samples as determined by SYBR-14 and PI staining after thawing and washing of the spermatozoa (three replicates, mean ± SEM). Sires are ranked according to increasing NRR

A positive association between the loge transformed number of spermatozoa bound to 0.1 mm2 of oviduct epithelium and the NRR was found after 24 h of coincubation and only when the membrane integrity of the initial sperm sample was >60% (P < 0.05; Table 3). Figure 4 shows the inverse predicted NRR and 95% confidence interval (CI) of a bull with a membrane integrity of 85%. For example, when a mean of 40 spermatozoa were bound to 0.1 mm2 of oviduct epithelium, the inverse predicted NRR of the bull is approximately 68% (95% CI: 61% to >70%).


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  		TABLE 3. Regression lines* of the number of spermatozoa bound to epithelial oviduct explants versus the NRR and different membrane integrity (MI) rates after 24 h of culture



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  		FIG. 4. Inverse prediction of the NRR (95% CI) of a sperm sample with a membrane integrity of 85% and the given number of spermatozoa bound to the epithelial oviduct explant. 40a, Number of spermatozoa/0.1 mm2 of oviduct explant. 68b, Inverse predicted point estimate of the NRR given the number of spermatozoa/0.1 mm2 of explant. The inverse predicted point estimate is obtained by using the intersection of the number of spermatozoa/0.1 mm2 with the fitted curve. 61c, Inverse predicted lower 95% prediction limit. The inverse predicted lower 95% prediction limit is obtained by using the intersection of the number of spermatozoa/0.1 mm2 of explant with the upper limit of the 95% CI of the fitted curve


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To study the binding of sperm to oviduct epithelial explants, it is important to choose the right incubation medium in which membranes of both oviduct cells and spermatozoa remain intact for a minimum of 48 h. This aspect was very important in our study, in contrast to other studies in which only short incubation periods were used [4, 20, 21]. In preliminary experiments, Hepes-TALP was as a more suitable medium for the survival of both spermatozoa and oviduct explants than were the other tested media (IVF-TALP, TCM-199 + 10% FCS, and TCM-199 alone). After 24 and 48 h of incubation, significantly more membrane-damaged and capacitated spermatozoa were observed in the other media than in Hepes-TALP (P < 0.05; data not shown). Furthermore, more spermatozoa survived while bound to oviduct explants in Hepes-TALP in the presence of oviduct epithelial cells. All tested media except Hepes-TALP were bicarbonate buffered; it is known that bicarbonate induces capacitation of spermatozoa [1, 22]. Capacitation appears to be involved in the release of bovine spermatozoa from oviduct epithelium [16] and in combination with decreased sperm survival is responsible for the important drop in sperm binding to oviduct explants after 24 h of incubation in IVF-TALP. TCM-199 with or without the addition of 10% FCS was useful only for the incubation of oviduct explants but not for incubation of spermatozoa (unpublished data). Only a few spermatozoa survived in TCM-199, which is why binding capacity was lower in this medium. One could argue that dead sperm could exert a negative influence on binding of remaining living spermatozoa to the oviduct explants by the release of enzymes or other substances [16]; however, this influence was not substantiated by a change in pH in any of the media. The tendency of frozen-thawed spermatozoa to have a lower binding capacity and shorter survival than fresh spermatozoa may be due to cell damage and the induction of capacitation-like changes during freezing and thawing procedures [23]. Moreover, capacitation is known to destabilize the sperm plasma membrane [1, 24] and thus reduce the life span of the sperm [25].

Sperm binding should be investigated with oviduct explants smaller than 20 000 µm2 to provide more repeatable results. Spermatozoa are not evenly distributed over the surface. They are spaced closely in some areas and sparsely in others and are absent in a few areas, as has been observed previously [16]. Sperm heads bind preferentially to the cilia or in deeper regions of ciliated epithelial cells between the cilia and not to secretory epithelial cells [12, 21]. Further experiments using electron microscopy are needed to determine whether oviduct explants with a small surface area consist of more ciliated epithelial cells and less secretory cells than do explants with a large surface area.

To investigate the binding density of bovine spermatozoa to oviduct epithelial explants, two very important criteria have to be met: sperm counting must be performed easily and repeatedly, and sperm binding must mimic the in vivo situation as closely as possible. The use of fluorescent dyes for the evaluation of sperm binding has two advantages. First, staining with JC-1, which labels the mitochondria of both spermatozoa and oviduct cells, makes it easy to count the tails of the spermatozoa bound to oviduct explants and to measure the surface area of the oviduct explant. Second, PI stains only membrane-damaged spermatozoa. In this way membrane-intact and membrane-damaged spermatozoa can be distinguished so that only membrane-intact spermatozoa are counted, which is not possible with videomicroscopy. Several methods for studying binding of sperm to oviduct epithelial cells in mammals have been described [15, 26]. Sperm binding density is usually evaluated on oviduct explants [4, 16, 20, 26, 27] or on oviduct monolayers [12, 15, 28–30]. In our study, oviduct explants were used as a model for oviduct epithelial cells in vivo because of the maintenance of most of the morphological characteristics [20]. Because morphological differentiation and polarization of epithelial cells is more pronounced in polarized explants, there is higher sperm binding density in explants than in epithelial culture monolayers, as shown in humans [31]. The number of spermatozoa bound to living or fixed cocultures has been counted by means of scanning electron microscopy analysis [12, 15], videomicroscopy, and image analysis [4, 16, 20] or by labeling spermatozoa with the fluorochrome Hoechst 33342 followed by counting attached spermatozoa by means of image processing and analysis of fluorescent video images [26]. Thomas et al. [26] were the first to develop a cytofluorescent assay for counting large numbers of labeled spermatozoa attached to somatic cell monolayers. However, the application of Hoechst 33342 leads to nuclear staining of both spermatozoa and oviduct explant cells, which makes it more difficult to distinguish spermatozoa from oviduct cells. This problem does not occur with JC-1. Although sperm heads and mitochondria in the oviduct cells also stain faintly positive, this staining does not interfere with the counting of sperm.

Linking in vivo fertility with a relatively fast and cheap in vitro evaluation method would be very valuable for the prediction of fertility of a sire. The standard procedure for evaluating the fertility of semen from sires is to determine pregnancy data following AI. This procedure is time consuming and expensive because of the large number of young bulls entering the breeding program [32]. Several studies have already been done to find a simple and reliable test for fertility [33]. Despite the fact that a number of studies have focused on single sperm traits such as sperm morphology [34], sperm motility [35, 36], and the presence of intact acrosomes [37], none of these traits were correlated significantly with in vivo fertility. Because fertilization requires several sperm activities, it would be better to combine different sperm traits to achieve a better correlation between in vitro tests and in vivo fertility [32, 38, 39]. With the in vitro model that we optimized, we established a correlation between density of sperm binding to oviduct explants and NRR. Spermatozoa that can bind to oviduct explants are characterized by an uncapacitated status [16], an intact acrosome [15], a superior morphology [29], and a normal chromatin structure [40]. Thundathil et al. [41] demonstrated that the proportion of uncapacitated spermatozoa present in frozen-thawed bull semen varies among bulls and, more important, that the presence of uncapacitated spermatozoa is positively correlated with fertility. Uncapacitated spermatozoa have an advantage over capacitated spermatozoa during their transit to the site of fertilization in the oviduct because they are more likely to survive. If capacitation were to occur before the spermatozoa reached the oviduct, the sperm population available for fertilization would be reduced, causing an adverse effect on fertilization. When the percentage of uncapacitated spermatozoa in a sperm sample is high, more spermatozoa are able to bind to oviduct explants, which may result in a higher fertility rate. Differences among individual animals in the capacity of sperm to bind to oviduct epithelium in vitro have already been reported for stallions and boars [21, 28]. However, in these studies no data were available on the comparative ranking of the fertility, so the relationship between sperm-oviduct binding index and fertility could not be established.

Using our in vitro model, we found that the number of spermatozoa bound to oviduct explants coincubated for 24 h is positively associated with in vivo fertility only when the membrane integrity of the initial sperm sample is >60%. No association between the number of spermatozoa bound to oviduct explants and NRR was found after 30 min and 48 h of coincubation and after 24 h of coincubation when the membrane integrity of the sperm sample was <60%.

The model can be used for predicting the NRR of bulls when sperm samples with membrane integrity rates >60% are coincubated with oviduct explants for 24 h. However, the width of the 95% CI is large and could be narrowed by collecting more data to predict NRR and by controlling for factors that can bias NRR, such as technician and age and parity of the cow. Moreover, testing of multiple ejaculates per bull could increase the accuracy of the model.

In the present study, labeling of both spermatozoa and oviduct explants with the fluorescent carbocyanine dye JC-1 in combination with PI without fixing provided a rapid, reliable, and reproducible method for counting the number of membrane-intact spermatozoa bound to living oviduct explants. The results of this analysis indicate that the capacity of spermatozoa bound to oviduct explants in vitro varies among bulls and that the number of spermatozoa bound to oviduct epithelial explants is positively correlated with the NRR after 24 h of coincubation when the membrane integrity of the initial sperm sample is >60%.


   ACKNOWLEDGMENTS
 
We thank the VRV (Flemish Cattle Breeding Association, Belgium) for supplying the batches of frozen semen and for providing the NRR. We also thank Dr. Coryn for critical reading, and K. Goeman, G. Spaepen, and J. Mestach for technical assistance.


   FOOTNOTES
 
1 This research was supported by the Ministry of Agriculture of the Belgian government (grant S6012), by the Flemish Cattle Breeding Association VRV, and by the Research Fund of Ghent University (grant 011B8698). Back

2 Correspondence: Ann Van Soom, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium. FAX: 32 9 264 77 97; ann.vansoom{at}rug.ac.be Back

Received: 25 November 2001.

First decision: 27 December 2001.

Accepted: 25 April 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill J (eds.), Physiology of Reproduction. New York: Raven Press; 1994: 189–317
  2. Hunter RHF, Wilmut I. The rate of functional sperm transport into the oviducts of mated cows. Anim Reprod Sci 1982 5:167-173
  3. Suarez SS, Drost M, Redfern K, Gottlieb W. Sperm motility in the oviduct. In: Serono Symposia, 1990; Norwell, MA; 111–124
  4. Lefebvre R, Lo MC, Suarez SS. Bovine sperm binding to oviductal epithelium involves fucose recognition. Biol Reprod 1997 56:1198-1204[Abstract]
  5. Suarez SS, Brockman K, Lefebvre R. Distribution of mucus and sperm in bovine oviducts after artificial insemination: the physical environment of the oviductal sperm reservoir. Biol Reprod 1997 56:447-453[Abstract]
  6. Hunter RHF. Sperm dynamics in the female genital tract: interactions with fallopian tube microenvironments. In: Motta PM (ed.), Microscopy of Reproduction and Development: A Dynamic Approach. Rome: Antonio Delfino Editore; 1997: 35–45
  7. Hunter RH, Wilmut I. Sperm transport in the cow: peri-ovulatory redistribution of viable cells within the oviduct. Reprod Nutr Dev 1984 24:597-608
  8. Day FT. Survival of sperm in the genital tract of the mare. J Agric Sci 1942 32:108-111
  9. Den Daas JHG, De Jong G, Lansbergen LMTE, Van Wagtendonk-De Leeuw AM. The relationship between the number of spermatozoa inseminated and the reproductive efficiency of individual dairy bulls. J Dairy Sci 1998 81:1714-1723[Abstract]
  10. Parker WG, Sullivan JJ, First NL. Sperm transport and distribution in the mare. J Reprod Fertil Suppl 1975 23:63-66
  11. Mburu JN, Einarsson S, Lundeheim N, Rodriguez-Martinez H. Distribution, number and membrane integrity of spermatozoa in the pig oviduct in relation to spontaneous ovulation. Anim Reprod Sci 1996 45:109-121[CrossRef][Medline]
  12. Pollard JW, Plante C, King WA, Hansen PJ, Betteridge KJ, Suarez SS. Fertilizing capacity of bovine sperm may be maintained by binding to oviductal epithelial cells. Biol Reprod 1991 44:102-107[Abstract]
  13. Bosch P, de Avila JM, Ellington JE, Wright RW Jr. Heparin and Ca2+-free medium can enhance release of bull sperm attached to oviductal epithelial cell monolayers. Theriogenology 2001 56:247-260[CrossRef][Medline]
  14. Talevi R, Gualtieri R. Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro. Biol Reprod 2001 64:491-498[Abstract/Free Full Text]
  15. Gualtieri R, Talevi R. In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release. Biol Reprod 2000 62:1754-1762[Abstract/Free Full Text]
  16. Lefebvre R, Suarez SS. Effect of capacitation on bull sperm binding to homologous oviductal epithelium. Biol Reprod 1996 54:575-582[Abstract]
  17. Madison V, Greve T, Avery B, Wamberg T. The effect of endotoxin-contaminated medium on in vitro fertilization and development of bovine oocytes matured in vitro. Reprod Nutr Dev 1991 32:159-165
  18. Garner DL, Thomas CA. Organelle-specific probe JC-1 identifies membrane potential differences in the mitochondrial function of bovine sperm. Mol Reprod Dev 1999 53:222-229[CrossRef][Medline]
  19. Garner DL, Pinkel D, Johnson LA, Pace MM. Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Biol Reprod 1986 34:127-138[Abstract]
  20. Lefebvre R, Chenoweth PJ, Drost M, LeClear CT, MacCubbin M, Dutton JT, Suarez SS. Characterization of the oviductal sperm reservoir in cattle. Biol Reprod 1995 53:1066-1074[Abstract]
  21. Petrunkina AM, Gehlhaar R, Drommer W, Waberski D, Töpfer-Petersen E. Selective sperm binding to pig oviductal epithelium in vitro. Reproduction 2001 121:889-896[Abstract]
  22. Harrison RA, Ashworth PJ, Miller NG. Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membrane. Mol Reprod Dev 1996 45:378-391[CrossRef][Medline]
  23. Cormier N, Sirard MA, Bailey JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Androl 1997 18:461-468[Abstract/Free Full Text]
  24. Langlais J, Roberts KD. A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 1985 12:183-224
  25. Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev 1995 7:871-891[CrossRef][Medline]
  26. Thomas PG, Ball BA, Brinsko SP. Interaction of equine spermatozoa with oviduct epithelial cell explants is affected by estrous cycle and anatomic origin of explant. Biol Reprod 1994 51:222-228[Abstract]
  27. Raychoudhury SS, Suarez SS. Porcine sperm binding to oviductal explants in culture. Theriogenology 1991 36:1059-1070[CrossRef]
  28. Thomas PG, Ball BA. Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro. Mol Reprod Dev 1996 43:55-61[CrossRef][Medline]
  29. Thomas PG, Ball BA, Miller PG, Brinsko SP, Southwood L. A subpopulation of morphologically normal, motile spermatozoa attach to equine oviductal epithelial cell monolayers. Biol Reprod 1994 51:303-309[Abstract]
  30. Ellington JE, Samper JC, Jones AE, Oliver SA, Burnett KM, Wright RW. In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products. Anim Reprod Sci 1999 56:51-65[CrossRef][Medline]
  31. Baillie HS, Pacey AA, Warren MA, Scudamore IW, Barratt CL. Greater numbers of human spermatozoa associate with endosalpingeal cells derived from the isthmus compared with those from the ampulla. Hum Reprod 1997 12:1985-1992[Abstract/Free Full Text]
  32. Zhang BR, Larsson B, Lundeheim N, Haard MG, Rodriguez-Martinez H. Prediction of bull fertility by combined in vitro assessments of frozen-thawed semen from young dairy bulls entering an AI-programme. Int J Androl 1999 22:253-260[CrossRef][Medline]
  33. Larsson B, Rodriguez-Martinez H. Can we use in vitro fertilization tests to predict semen fertility?. Anim Reprod Sci 2000 60:327-336
  34. Barth AD. Factors affecting fertility with artificial insemination. Vet Clin North Am Food Anim Pract 1993 9:275-289[Medline]
  35. Stalhammar EM, Janson L, Philipsson J. The impact of sperm motility on non-return rate in preselected dairy bulls. Reprod Nutr Dev 1994 34:37-45
  36. Holt C, Holt WV, Moore HD, Reed HC, Curnock RM. Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials. J Androl 1997 18:312-323[Abstract/Free Full Text]
  37. Cumming IR. Suitability of the intact acrosome method for the prediction of fertility in bovine artificial insemination. Vet Rec 1995 136:289-291[Abstract]
  38. Amann RP, Hammerstedt RH. In vitro evaluation of sperm quality: an opinion. J Androl 1993 14:397-406[Free Full Text]
  39. Farrell PB, Presicce GA, Brockett CC, Foote RH. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 1998 49:871-879[CrossRef][Medline]
  40. Ellington JE, Evenson DP, Wright RW Jr, Jones AE, Schneider CS, Hiss GA, Brisbois RS. Higher quality human sperm in a sample selectively attach to oviduct (Fallopian tube) epithelial cells in vitro. Fertil Steril 1999 71:924-929[CrossRef][Medline]
  41. Thundathil J, Gil J, Januskauskas A, Larrson B, Soderquist L, Mapletoft R, Rodriguez-Martinez H. Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination. Int J Androl 1999 22:366-373[CrossRef][Medline]



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