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Migration of bovine spermatozoa in a synthetic medium and its relation to in vivo bull fertility
Theriogenology 58 (2002) 1027±1037
Migration of bovine spermatozoa in a synthetic medium and its relation to in vivo bull fertility S. Verberckmoes*, A. Van Soom, I. De Pauw, J. Dewulf, A. de Kruif Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 133 Salisburylaan, 9820 Merelbeke, Belgium Received 19 September 2001; accepted 19 February 2002
Abstract Although sperm migration has been extensively re®ned and validated in human infertility studies, its application to predict bovine fertility has been very limited, and a clear relation between the sperm migration distance and in vivo bull fertility has never been demonstrated. A synthetic medium based upon methyl cellulose (MC) was tested for its suitability to serve as a migration medium for frozenthawed bovine spermatozoa. The effects of the concentration of MC, the incubation time, and sperm concentration on sperm migration capacity was determined. The relation between sperm migration capacity at different incubation times of the frozen-thawed spermatozoa of ®ve bulls, and their 56 days nonreturn rates (NRRs) was assessed in order to evaluate its suitability as a tool to predict in vivo bull fertility. The highest repeatability of the sperm migration test (CV 10:7%) was obtained when the sperm migration distance of the ®ve vanguard motile spermatozoa was determined at 30 min incubation at 37 8C in a migration medium with 1.35% MC. No signi®cant difference in migration distance was demonstrated when sperm concentrations of 100 106 and 150 106 spermatozoa/ml, respectively, were used. Despite the relatively high repeatability of the migration test, no relation was found between the sperm migration distance and the 56 days NRRs of ®ve sire bulls. Therefore, the sperm migration test in 1.35% MC cannot be used to predict in vivo bull fertility accurately. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Spermatozoa; Bovine; Fertility; Migration
1. Introduction During natural mating, spermatozoa must migrate through the cervical mucus to enter the uterus. Subsequently, spermatozoa are transported mainly by muscular movements of * Corresponding author. Tel.: 32-9-264-7564; fax: 32-9-264-7534. E-mail address: [email protected] (S. Verberckmoes).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 9 3 1 - 7
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the uterus to the functional sperm reservoir in the oviduct [1]. It is obvious that when spermatozoa cannot reach the site of fertilization for any reason, they will be unable to reach their potential to fertilize [2,3]. To study spermatozoal motility potential, physical barriers to be overcome in vivo can be mimicked by cervical mucus in sperm migration assays. Cervical mucus from human females [4] and from cows [5] has been used for sperm migration assays to evaluate the fertility of human semen donors in vitro. In humans, a number of independent studies have shown that the fertilizing capacity of spermatozoa is correlated with penetration of cervical mucus [6,7]. However, sperm migration distances are in¯uenced by the mucus donor and by the length of time the mucus was stored before testing [5,8]. In addition, there might be technical dif®culties with ®lling capillary tubes with cervical mucus [9]. The use of a substitute for cervical mucus can overcome the problem of characteristic changes in the cervical mucus over time [10]. Alternatives that have been tested in sperm migration assays are hen egg white [11±13], hyaluronic acid [14,15,10] and polyacrylamide gel [16±19]. Although sperm migration has been extensively re®ned and validated in human infertility studies [20,21], its application to predict bovine fertility has been very limited and a clear relation between the sperm migration distance and in vivo bull fertility has never been demonstrated [5,22]. The aim of this study was to test a simple sperm migration assay for cattle, which can be performed easily under ®eld conditions. For this purpose, a synthetic medium based upon methyl cellulose (MC) was evaluated for its suitability as a sperm migration medium. Methyl cellulose is an unbranched chain polymer and has been extensively used to test effects of viscosity on sperm movements [23,24]. The repeatability of the sperm migration test was optimised and the relation between bovine fertility as assessed by 56 days nonreturn rates (NRRs) and the migration distance in this synthetic medium was investigated. 2. Material and methods 2.1. General material and methods 2.1.1. Synthetic migration medium The synthetic media consisted of a mixture of Hepes-TALP and MC of 1500 centipoise (cps) (Federa1, Brussels, Belgium). The ®nal solutions contained 0.27, 0.68 and 1.35% MC. Solutions were homogenized by shaking, and stored at 4 8C up to 1 week before use. The pH, osmolarity and viscosity of the three media were determined. Their pH was determined at 20 8C by means of a pH meter (pH 526, TUV1, Weilheim, Germany) and their osmolarity was assessed by determining the freezing point with an osmometer (Fisk Associates1, MA, USA). The viscosity of the media was determined at 37 8C by means of the Haake PG 142 viscometer (Haake1, Karlsruhe, Germany) at four rotation speeds: 64, 128, 256 and 512 rpm (Table 1). 2.1.2. Treatment of spermatozoa Frozen bull semen was provided by the Flemish Arti®cial Insemination Centre (VRV). The ejaculates were diluted in an egg yolk±Tris±glycerol extender. The semen was frozen
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Table 1 pH, osmolarity and viscosity of migration medium 1, 2 and 3 with 0.27, 0.68 and 1.35% MC, respectively, determined at four rotation speeds Medium
1 2 3
Concentration of MC (%)
0.27 0.68 1.35
pH
7.1 7.1 7.1
Osmolarity
295 289 287
Viscosity (mPa s) 64 rpm
128 rpm
256 rpm
512 rpm
6.8 16.5 65.5
5.4 13.4 53.0
4.5 11.6 53.0
4.9 9.8 31.4
in 0.25 ml straws and maintained in liquid nitrogen at 196 8C until use. Frozen semen of the same batch was used throughout the experiment. To determine the repeatability of the sperm migration assay, frozen semen of six bulls was used. The frozen-thawed semen used for the determination of the NRRs originated from the same batch as the semen used for the sperm migration test. 2.1.3. Sperm migration test The in vitro migration apparatus consisted of nonheparinised round microhaematocrite capillaries (Hirschmann1, Eberstadt, Germany) which were ®xed in plastic petridishes to facilitate handling. In combination with inverted microscopy, round capillaries are as suitable as specially designed ¯at tubes [5]. The length of the capillaries was 7.5 cm and they had an internal diameter of 1.5 mm. They were ®lled with the selected migration medium and closed at one end with putty. At the other end, 10 ml of space was left in which the sperm suspension was injected. The capillaries were prewarmed in a humidi®ed chamber at 37 8C. Only capillaries free of air bubbles were used. At time zero (t0), 10 ml of sperm suspension was injected in the open end of the capillary. The migration distance was determined at different time intervals after injection by means of an inverted light microscope (Olympus1 IX70-S8F, Japan) equipped with a heating stage at 37 8C at magni®cation of 200 (Fig. 1). The capillaries were examined microscopically from the end where the sperm was injected to the most distant point containing motile spermatozoa. Since we were interested in spermatozoa with a good migration capacity, the `migration distance' for the vanguard one and ®ve spermatozoa (including the vanguard one) was measured (millimetres). The migration distance of the vanguard spermatozoa was measured from the place of injection to the farthest point of migration. 2.2. Experimental design and statistical analysis 2.2.1. Experiment 1 In¯uence of MC concentration on the sperm migration distance. In this experiment, for each concentration of MC (0.27, 0.68 and 1.35% MC) six replicates per bull were used. The test was performed with frozen-thawed semen of six bulls. After thawing the semen, 1 ml of Hepes-TALP was added and the sperm suspension was centrifuged for 5 min at 514 g. The supernatant was removed after centrifugation and the spermatozoa were diluted to obtain a ®nal concentration of 120 106 spermatozoa/ml. Sperm quality was determined before injection into the capillary tubes for the sperm migration test. The migration
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Fig. 1. Determination of sperm migration distance in vitro by means round microhaematocrite capillaries, which were ®xed in plastic petridishes to facilitate handling, in combination with inverted microscopy.
distance was determined for the one and ®ve vanguard motile spermatozoa at 15 and 30 min after injection of the spermatozoa into the capillary tubes and incubation in the humidi®ed chamber at 37 8C. The comparison of the different media and different spermatozoa concentrations in relation to the sperm migration distance was done by using ANOVA (SPSS 10, SPSS Inc., IL 60611, USA, 1999). 2.2.2. Experiment 2 Repeatability of the sperm migration test. Semen from six bulls was used to measure migration in 1.35% MC. Per bull, six replicates of the sperm migration test were performed. In each capillary 120 106 spermatozoa/ml of spermatozoa were injected and the migration distance of the one and ®ve vanguard motile spermatozoa was determined at 15 (t1), 30 (t2), 45 (t3) and 60 min (t4) after incubation in the humidi®ed chamber at 37 8C. The repeatability of the sperm migration test was determined by evaluating the coef®cient of variation (CV) for the one and ®ve vanguard motile spermatozoa. 2.2.3. Experiment 3 In¯uence of sperm concentration on the sperm migration distance. Semen from two bulls was used to perform the sperm migration assay in a medium with 1.35% MC. The frozenthawed semen was centrifuged for 5 min at 514 g and concentrated to 100 106 and 150 106 spermatozoa/ml. For each concentration, six replicates per bull were tested. In contrast to the previous experiments, the migration distance was not determined by assessing the migration point of the one and ®ve vanguard spermatozoa but was determined by the most distant point containing ®ve motile spermatozoa per microscopic ®eld at t0 (moment of injection), t1 (15 min) and t2 (30 min) after injection of the spermatozoa. The in¯uence of the two sperm concentrations (100 106 and 150 106 spermatozoa/ml) on the sperm migration distance was determined by means of the independent sample t-test (SPSS 10, SPSS Inc., IL 60611, USA, 1999).
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2.2.4. Experiment 4 Relation between four sperm quality parameters and their predictive value for NRRs. Frozen-thawed semen from ®ve bulls with NRRs ranging from 52.8 to 66.7% was used to determine progressive motility, membrane integrity, sperm concentration and the sperm migration distance. The spermatozoa used for the sperm migration assay were of the same batch as those used for the determination of in vivo fertility. The NRRs were determined after 219 16 (mean S:D:) ®rst inseminations in heifers and cows. The inseminations were performed 12±18 h after the ®rst estrous signs observed by the farmer. The NRRs were not known by the investigators at the time the sperm migration experiments were performed. Prior to freezing, the semen from the ®ve bulls exhibited minimum values of 80% progressive motility, 80% membrane intact spermatozoa and a concentration of at least 0:75 109 spermatozoa/ml. After thawing, the percentage of progressive motile and membrane intact spermatozoa and sperm concentration ranged from 25±50 to 62±86% and 6 106 to 14 106 spermatozoa/ml, respectively, for the ®ve bulls. The progressive motility of the bull semen was evaluated subjectively at 37 8C. Eosin± nigrosin staining was used for the assessment of membrane integrity. The sperm concentration was determined by means of a BuÈrker counting chamber. These three basic parameters were evaluated by means of light microscopy. The sperm migration capacity was determined in the Hepes-TALP medium containing 1.35% MC. Six replicates of the sperm migration assay were performed per bull. The migration distance of the ®ve vanguard spermatozoa was measured at 15 min (t1) and 30 min (t2) after injecting 120 106 spermatozoa/ml. The correlation between the sperm migration distance at t2 and the three other sperm quality parameters, as well as the correlation between the NRRs of the ®ve bulls, and the progressive motility, membrane integrity and sperm migration distance at t2, was determined by calculating the correlation coef®cient (r). Linear regression was used to evaluate the predictive properties of the four sperm quality parameters on in vivo fertility (NRR), as well as to evaluate the relation between the sperm migration distance and the three other sperm quality parameters (SPSS 10, SPSS Inc., IL 60611, USA, 1999). 3. Results 3.1. Experiment 1: In¯uence of MC concentration on the sperm migration distance The migration distance of the one and ®ve vanguard spermatozoa after 15 min of incubation was signi®cantly shorter in the medium with the highest viscosity than in the two other media. After 30 min of incubation, the migration distance for the one and ®ve vanguard motile spermatozoa was signi®cantly different for the three media (P < 0:05) (Table 2). The most viscous medium (1.35% MC) yielded the lowest mean sperm migration distance. 3.2. Experiment 2: Repeatability of the sperm migration test From Experiment 2, only medium 3 was used for the sperm migration assay. The repeatability of the sperm migration assay in the medium with 1.35% MC for all six bulls is
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Table 2 Migration distance in mm (mean S:E:M:) for six replicates per bull (n 6) of the vanguard one and five motile spermatozoa after 15 and 30 min incubation in migration medium with three different concentrations of MC Medium
Migration distance at 15 min Vanguard one
0.27% MC 0.68% MC 1.35% MC
Migration distance at 30 min
Vanguard five
a
a
30.9 0.23 28.6 0.23a 18.3 0.23b
26.8 0.19 23.6 0.19a 16.0 0.19b
Vanguard one c
44.9 0.27 38.3 0.27d 25.7 0.27e
Vanguard five 40.4 0.26c 32.8 0.26d 21.4 0.26e
Values with different superscripts (a, b, c, d, e) in the same column are signi®cantly different (P < 0:05).
presented in Table 3. The repeatability of the sperm migration assay increased with increasing incubation time and was higher for the ®ve vanguard spermatozoa than for the one vanguard spermatozoon. It was noticed that the number of motile spermatozoa decreased substantially after >30 min incubation at 37 8C. Therefore, the sperm migration at a later point of time was not considered reliable despite the low CV. From Table 3, it is clear that the highest repeatability at t2 is obtained for the ®ve vanguard spermatozoa (CV 10:7 3:7). 3.3. Experiment 3: In¯uence of sperm concentration on the sperm migration distance Experiment 3, although the ®ve vanguard spermatozoa at t1 and t2 of incubation showed a tendency to migrate more at a concentration of 150 106 spermatozoa/ml than at a concentration of 100 106 spermatozoa/ml, this difference was only signi®cant at t1 (P 0:89 at t0, P 0:02 at t1, P 0:06 at t2) (Fig. 2). 3.4. Experiment 4: Relation between four different sperm quality parameters and their predictive value for NRRs Experiment 4 as is shown in Table 4, sperm concentration (P < 0:01) and membrane integrity (P 0:05) were signi®cantly related to the sperm migration distance at t2. Moreover, the sperm concentration could explain 64% of the variation in migration Table 3 Repeatability of the sperm migration assay determined by the CV for the vanguard one and five motile spermatozoa in migration medium with 1.35% MC Time of incubation (min) 15 30 45 60
(t1) (t2) (t3) (t4)
Average
Coefficient of variation Vanguard one spermatozoon
Vanguard five spermatozoa
11.4 11.0 10.0 8.1
11.1 10.7 7.5 5.4
10.0
8.5
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Fig. 2. In¯uence of sperm concentration on sperm migration distance at 0, 15 and 30 min after injection of spermatozoa in the capillaries. Table 4 Relation between progressive motility, membrane integrity, sperm concentration and sperm migration distance of bovine spermatozoa was tested at 30 min of incubation in a migration medium of 1.35% methyl cellulose Sperm migration distance at t2 b0 Progressive motility Membrane integrity Concentration
36.48 26.27 22.00
b1 0.02 0.14 0.15
P
r
0.81 0.05 <0.01
0.05 0.36 0.64
Linear regression was used to determine the regression coef®cients (b0 and b1) and the correlation coef®cient (r).
distance. The correlation between progressive motility and the sperm migration distance was very low (r 0:05). As is presented in Table 5, it is obvious that the progressive motility was the sperm quality parameter which is most correlated (r 0:66, P < 0:01) to the NRRs of the ®ve bulls. The sperm migration distance at t1 and t2 was also signi®cantly related to the NRRs, albeit in a negative way (b1 0:50 and 0.43, respectively). The correlation between membrane integrity and NRRs was negligible (r 0:05). Table 5 Use of sperm quality parameters: progressive motility, membrane integrity, migration distance after 15 min (t1) and 30 min (t2) incubation in a medium of 1.35% methyl cellulose to predict NRR NRR b0 Progressive motility Membrane integrity Migration distance at t1 Migration distance at t2
49.32 58.19 74.75 74.69
b1 0.32 0.02 0.50 0.43
P
r
<0.01 0.81 <0.01 0.02
0.66 0.05 0.52 0.43
Linear regression was used to determine the regression coef®cients (b0 and b1) and the correlation coef®cient (r).
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4. Discussion The capacity for sperm migration through cervical mucus depends on the structural± mechanical properties of the mucus microstructure, the viscosity of the mucus plasma and the size and shape of the sperm head and ¯agellum [3]. In this study, we used a synthetic medium based upon MC instead of cervical mucus to obtain a sperm migration medium that could be tested for its suitability to predict in vivo bull fertility. Cervical mucus of estrous cattle has a pH of about 7, a viscosity of 23.2 mm H2O, and a spinnbarkeit of 72 mm. Spermatozoa can migrate in cervical mucus on average a distance of 13 mm for the ®rst 10 min [25]. The migration medium with the highest concentration of MC (1.35% MC) had a pH of 7.1, and spermatozoa migrated a distance of 16 mm within 15 min, which is very similar to the values obtained in bovine cervical mucus. However, the spinnbarkeit of our MC medium was only 7 mm, and unlike polyacrylamide or cervical mucus, it did not have the appearance of a gel. The 1.35% MC medium had a viscosity of 65.5 mPa s but because of the difference in units, it is dif®cult to compare the viscosity of the MC medium with that of cervical mucus. From Experiment 1, it is obvious that sperm movement was depressed when the viscosity of the sperm migration medium increased. Since the sperm migration capacity in the 1.35% MC medium and cervical mucus were relatively similar, and the length of the capillary tubes was not a limiting factor for sperm migration capacity in the 1.35% MC medium, this medium was used for further experiments. Methyl cellulose is an unstructured and unreactive buffer that interferes with sperm migration only because of its increased viscosity and was, therefore, perfectly suited for our purpose, which was to evaluate sperm migration through a synthetic medium with good repeatability. Sperm migration in a MC solution was quite repeatable as indicated by a CV of 10.7%. Sperm samples which were introduced into the capillary at two different concentrations showed a tendency to migrate further at a concentration of 150 106 spermatozoa/ml than at a concentration of 100 106 spermatozoa/ml, although this was only signi®cant at t1 (P 0:02). This tendency was con®rmed in Experiment 4, where concentration had a signi®cant positive correlation with the sperm migration distance. In a number of studies, sperm migration capacity has been linked with other sperm characteristics such as total motility, sperm morphology, progressive motility, acrosome integrity and sperm concentration [7,26]. In our study, only the correlations between sperm migration capacity and progressive motility, membrane integrity and sperm concentration were determined. In a recent study, Anilkumar et al. [27] found that sperm penetration in polyacrylamide gel and bovine cervical mucus was positively correlated with post-thaw motility (r 0:81; r 0:89: P < 0:01) and acrosome integrity (r 0:88; r 0:94: P < 0:01) of bovine spermatozoa. However, in contrast to this [27] and other studies [17,26], in our study no correlation was found between progressive motility and the sperm migration distance (r 0:05, P 0:81). In accordance with Galli et al. [26] and Goldstein et al. [17], we did found a positive correlation between the sperm migration distance and membrane integrity (r 0:36, P 0:05) and concentration (r 0:64, P < 0:01). Although sperm migration capacity has been linked with different sperm quality parameters, it has seldom been linked with in vivo fertility in ruminants [28]. In contrast to studies in humans [6,17], we were not able to establish a positive correlation between the sperm migration distance in a migration medium with 1.35% MC and the NRRs of ®ve
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proven sires. The sperm migration distance at t1 and t2 was even negatively related (b1 0:50 and 0.43, respectively; P < 0:01 and 0.02, respectively) to the NRRs. In contrast to this, bull fertility and the sperm migration distance were linked in a positive way in a previous study [28]. However, the experimental design in that study differed from ours in two ways: ®rst, the sperm migration assay was performed in cervical mucus, and second, fresh spermatozoa were used, whereas the NRRs were obtained after insemination with frozen-thawed spermatozoa. This means that different ejaculates were used for the sperm migration assay and for the determination of the NRRs, whereas in our study the frozenthawed semen used for the sperm migration test and for the determination of the NRRs originated from the same batch. Apart from the experimental design, there are several explanations that may explain the disparity in results between different bovine studies and between bovine and human experiments. First, it could be due to differences in characteristics between the media used. Despite the fact that the MC medium yielded repeatable results it might not be as effective as hyaluronate, cervical mucus or polyacrylamide gel for sperm migration tests. Hyaluronate and cervical mucus possess a particular alignment of mucin chains resulting in a higher viscosity and in sperm movement parallel to the axis of the tube into mucus and hyaluronate solution channels [9]. In contrast to cervical mucus, the sperm penetrability in synthetic media such as MC and hyaluronate is not impaired by the presence of anti-sperm antibodies [9]. Polyacrylamide has proved to be more effective than MC when it comes to favouring migration of hyperactivated spermatozoa, at least in hamsters [24]. Whether this property of the medium is really important for studying bovine sperm migration remains to be determined, since it is known that bull spermatozoa are not hyperactivated at ejaculation, although they still have to penetrate the cervical mucus at this point of time [24]. Despite the fact that frozen-thawed bull spermatozoa are prone to premature capacitation [29], they do not hyperactivate as readily as hamster or mouse spermatozoa [24,30], and so it might be dif®cult to ®nd a relation between sperm migration and fertility in bulls, even if a more suitable medium such as polyacrylamide gel was used. Second, in contrast to humans, in cattle the difference in sperm quality between bulls is a lot smaller and the number of individuals with very poor sperm quality is very low. For the evaluation of the sperm migration test in humans, spermatozoa of fertile men (donors of an AI program) were compared to spermatozoa of patients attending an infertility laboratory [6,31]. Whereas in cattle the sperm migration test was performed with spermatozoa of bulls used for arti®cial insemination. These bulls have a high fertility resulting in a narrow range of NRRs, and therefore, the number of individuals with poor sperm quality in cattle is low [5]. In the only other comparable study of cattle, a relation was found between fertility and sperm migration, but interestingly, in this study one bull with 50% NRR and one with 14% NRR were compared with 11 bulls with NRRs ranging from 60 to 70% [28]. If the two bulls with the lowest NRRs were not included in the experiment, no relation could be found between NRR and the sperm migration distance. Even in the most carefully designed experiment in terms of examining the same ejaculate for sperm migration as for NRRs, sperm migration would probably only discriminate between bulls with extremely varying NRRs. Finally, the absence of a relation between sperm migration capacity and bull fertility could be explained by the fact that in dairy cattle, NRRs are determined after arti®cial insemination. During insemination the cervical barrier that spermatozoa have to overcome
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after natural mating is bypassed and the semen is deposited in the uterine body or in the uterine horns. It would be more logical to correlate sperm migration tests with fertility data obtained after natural mating, because then the spermatozoa have to migrate through the mucus before they reach the site of fertilization. It has been shown that sperm migration through cervical mucus in vitro could be indicative of the capacity of spermatozoa to populate the oviduct of goats [32]. After semen deposition at the external cervical os, spermatozoa from males with a high migration ef®ciency populated the oviduct more effectively than that from males with low migration ef®ciency, however differences were not signi®cant. However, this relation was not re¯ected in in vitro fertilization rates [32]. This may imply that capillary sperm migration tests may have value in predicting fertility under in vivo conditions, because the test is able to select superior sperm samples. In conclusion, we have shown that sperm migration through MC, albeit a repeatable test, could not be linked with in vivo bull fertility. Sperm migration through a viscous medium is only a very limited aspect of sperm behaviour that has to be performed in preparation to fertilize the oocyte. To obtain more precise prediction of in vivo fertility, it is imperative to combine different in vitro test systems as had been proposed by several authors [33,34]. Acknowledgements This research was supported by the Belgian Ministry of Agriculture, Grant no. S5867 and by the research fund of Ghent University, Grant no. 011B8698. References [1] Suarez SS, Drost M, Redfern K, Gottlieb W. Sperm motility in the oviduct. In: Bavister BD, Cummins J, Roland ERS, editors. Fertilization in mammals. Norwell MA: Serono Symposia, 1990. p. 111±24. [2] Hunter RHF. Human fertilization in vivo, with special reference to progression, storage and release of competent spermatozoa. Hum Reprod 1987;2:329±32. [3] Katz DF, Drobnis EZ, Overstreet JW. Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamet Res 1989;22:443±69. [4] Reichmann J, Insler V, Serr DV. A modi®ed in vitro spermatozoal penetration test. Int J Fertil 1973;18:241±5. [5] Kummerfeld HL, Lorton SP, Foote RH. Relationship of spermatozoal migration in cervical mucus to bovine fertility. J Androl 1981;2:103±7. [6] Alexander NJ. Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril 1981;36:201±8. [7] Hull MGR, McLeod FN, Joyce DN, Ray BD, McDermott A. Human in vitro fertilization, in vivo sperm penetration of cervical mucus and unexplained infertility. Lancet 1984;2:245±8. [8] Lee WI, Gaddum-Rosse P, Blandau RJ. Sperm penetration into cervical mucus in vitro. Part III. Effect of freezing on estrous bovine cervical mucus. Fertil Steril 1981;36:209±13. [9] Tang S, Garrett C, Gordon Baker HW. Comparison of human cervical mucus and arti®cial sperm penetration media. Hum Reprod 1999;14:2812±7. [10] Aitken RJ, Bowie H, Buckingham D, Harkiss D, Richardson DW, West KM. Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence. J Androl 1992;13:44±54. [11] Eggert-Kruse W, Gerhard I, Tilgen W, Runnebaum B. The use of hens egg white as a substitute for human cervical mucus in assessing human infertility. Int J Androl 1990;13:258±66.
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[12] Bostofte E, Bagger P, Michael A, Stakemann G. The sperm penetration test (P-test) can predict fecundability in the male partner from infertile couples. Andrologia 1992;24:124±9. [13] Engel S, Petzoldt R. Human sperm penetration in different media. Andrologia 1999;31:233±9. [14] Neuwinger J, Cooper TG, Knuth UA, Nieschlag E. Hyaluronic acid as medium for human sperm migration tests. Hum Reprod 1991;6:396±400. [15] Mortimer D, Mortimer ST, Shu MA, Swart R. A simpli®ed approach to sperm±cervical mucus interaction testing using a hyaluronate migration test. Hum Reprod 1990;5:835±41. [16] Lorton SP, Kummerfelt HT, Foote RH. Polyacrylamide as substitute for cervical mucus in sperm migration studies. Fertil Steril 1981;35:222±5. [17] Goldstein MC, Wix LS, Foote RH, Feldschuh R, Feldschuh J. Migration of fresh and cryopreserved human spermatozoa in polyacrylamide gel. Fertil Steril 1982;37:668±73. [18] Parrish JJ, Foote RH. Quanti®cation of bovine sperm separation by a swim up method. relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility. J Androl 1987;8:259±66. [19] Eggert-Kruse W, Schwalbach B, Rohr G, Klinga K, Tilgen W, Runnebaum B. Evaluation of polyacrylamide gel as substitute for human cervical mucus in the sperm penetration test. Fertil Steril 1993;60:540±9. [20] Clarke GN, Garrett C, Baker GHW. Quantitative sperm mucus penetration: modi®ed formulae for calculating penetration ef®ciency. Hum Reprod 1998;13:1255±9. [21] Biljan MM, Taylor CT, Manasse PA, Joughin EC, Kingsland CR, Lewis-Jones DI. Evaluation of different sperm function tests as screening methods for male fertilization potentialÐthe value of the sperm migration test. Fertil Steril 1994;62:591±8. [22] Matousek J, Riha J, Srsen V, Veselsky L, Louda F. Penetration of cervical mucus and other body ¯uids by bull sperm in capillary tubes. Anim Reprod Sci 1989;18:161±6. [23] Pate EF, Brokaw CJ. Movement of spermatozoa in viscous environments. J Exp Biol 1980;88:395±7. [24] Suarez SS, Dai X. Hyperactivation enhances mouse sperm capacity for penetrating viscoelastic media. Biol Reprod 1992;46:686±91. [25] Tsiligianni T, Karagiannidis A, Brikas P, Saratsis P. Physical properties of bovine cervical mucus during normal and induced (progesterone and/or PGF2a) estrus. Theriogenology 2001;55:629±40. [26] Galli A, Basetti W, Baldizzi D, Martignoni M, Bornaghi V, Maf® M. Frozen bovine semen quality and bovine cervical mucus penetration test. Theriogenology 1991;35:837±44. [27] Anilkumar R, Devanathan TG, Pattabiraman SR, Edwin MJ. Correlation between spermatozoal characteristics and sperm penetration distance in polyacrylamide gel and bovine cervical mucus. Theriogenology 2001;55:685±91. [28] Murase T, Okunda K, Sato K. Assessment of bull fertility using a mucus penetration test and a human chorionic gonadotrophin stimulation test. Theriogenology 1990;34:801±12. [29] Cormier N, Sirard MA, Bailey JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Androl 1997;18:461±8. [30] Chamberland A, Fournier V, Tardif S, Sirard MA, Sullivan R, Bailey JL. The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation. Theriogenology 2001;55: 823±35. [31] Insler V, Bernstein D, Glezerman M, Misgav N. Correlation of seminal ¯uid analysis with mucus penetrating ability of spermatozoa. Fertil Steril 1979;32:316±9. [32] Cox JF, Martinez C, Lagos S, Saravia F, Sasmay R. Sperm migration in cervical mucus in goats. Part II. Relationship with colonization of the oviduct and fertilization ef®ciency. Theriogenology 1997;47:254 [Abstract]. [33] Zhang BR, Larsson B, Lundeheim N, HaÊaÊrd MGH, Rodriguez-Martinez H. Prediction of bull fertility by combined in vitro assessments of frozen-thawed semen for young dairy bulls entering an AI-program. Int J Androl 1999;22:253±60. [34] Larsson B, Rodriguez-Martinez H. Can we use in vitro fertilization tests to predict semen fertility? Anim Reprod Sci 2000;60/61:327±36.
Migration of bovine spermatozoa in a synthetic medium and its relation to in vivo bull fertility S. Verberckmoes*, A. Van Soom, I. De Pauw, J. Dewulf, A. de Kruif Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 133 Salisburylaan, 9820 Merelbeke, Belgium Received 19 September 2001; accepted 19 February 2002
Abstract Although sperm migration has been extensively re®ned and validated in human infertility studies, its application to predict bovine fertility has been very limited, and a clear relation between the sperm migration distance and in vivo bull fertility has never been demonstrated. A synthetic medium based upon methyl cellulose (MC) was tested for its suitability to serve as a migration medium for frozenthawed bovine spermatozoa. The effects of the concentration of MC, the incubation time, and sperm concentration on sperm migration capacity was determined. The relation between sperm migration capacity at different incubation times of the frozen-thawed spermatozoa of ®ve bulls, and their 56 days nonreturn rates (NRRs) was assessed in order to evaluate its suitability as a tool to predict in vivo bull fertility. The highest repeatability of the sperm migration test (CV 10:7%) was obtained when the sperm migration distance of the ®ve vanguard motile spermatozoa was determined at 30 min incubation at 37 8C in a migration medium with 1.35% MC. No signi®cant difference in migration distance was demonstrated when sperm concentrations of 100 106 and 150 106 spermatozoa/ml, respectively, were used. Despite the relatively high repeatability of the migration test, no relation was found between the sperm migration distance and the 56 days NRRs of ®ve sire bulls. Therefore, the sperm migration test in 1.35% MC cannot be used to predict in vivo bull fertility accurately. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Spermatozoa; Bovine; Fertility; Migration
1. Introduction During natural mating, spermatozoa must migrate through the cervical mucus to enter the uterus. Subsequently, spermatozoa are transported mainly by muscular movements of * Corresponding author. Tel.: 32-9-264-7564; fax: 32-9-264-7534. E-mail address: [email protected] (S. Verberckmoes).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 9 3 1 - 7
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the uterus to the functional sperm reservoir in the oviduct [1]. It is obvious that when spermatozoa cannot reach the site of fertilization for any reason, they will be unable to reach their potential to fertilize [2,3]. To study spermatozoal motility potential, physical barriers to be overcome in vivo can be mimicked by cervical mucus in sperm migration assays. Cervical mucus from human females [4] and from cows [5] has been used for sperm migration assays to evaluate the fertility of human semen donors in vitro. In humans, a number of independent studies have shown that the fertilizing capacity of spermatozoa is correlated with penetration of cervical mucus [6,7]. However, sperm migration distances are in¯uenced by the mucus donor and by the length of time the mucus was stored before testing [5,8]. In addition, there might be technical dif®culties with ®lling capillary tubes with cervical mucus [9]. The use of a substitute for cervical mucus can overcome the problem of characteristic changes in the cervical mucus over time [10]. Alternatives that have been tested in sperm migration assays are hen egg white [11±13], hyaluronic acid [14,15,10] and polyacrylamide gel [16±19]. Although sperm migration has been extensively re®ned and validated in human infertility studies [20,21], its application to predict bovine fertility has been very limited and a clear relation between the sperm migration distance and in vivo bull fertility has never been demonstrated [5,22]. The aim of this study was to test a simple sperm migration assay for cattle, which can be performed easily under ®eld conditions. For this purpose, a synthetic medium based upon methyl cellulose (MC) was evaluated for its suitability as a sperm migration medium. Methyl cellulose is an unbranched chain polymer and has been extensively used to test effects of viscosity on sperm movements [23,24]. The repeatability of the sperm migration test was optimised and the relation between bovine fertility as assessed by 56 days nonreturn rates (NRRs) and the migration distance in this synthetic medium was investigated. 2. Material and methods 2.1. General material and methods 2.1.1. Synthetic migration medium The synthetic media consisted of a mixture of Hepes-TALP and MC of 1500 centipoise (cps) (Federa1, Brussels, Belgium). The ®nal solutions contained 0.27, 0.68 and 1.35% MC. Solutions were homogenized by shaking, and stored at 4 8C up to 1 week before use. The pH, osmolarity and viscosity of the three media were determined. Their pH was determined at 20 8C by means of a pH meter (pH 526, TUV1, Weilheim, Germany) and their osmolarity was assessed by determining the freezing point with an osmometer (Fisk Associates1, MA, USA). The viscosity of the media was determined at 37 8C by means of the Haake PG 142 viscometer (Haake1, Karlsruhe, Germany) at four rotation speeds: 64, 128, 256 and 512 rpm (Table 1). 2.1.2. Treatment of spermatozoa Frozen bull semen was provided by the Flemish Arti®cial Insemination Centre (VRV). The ejaculates were diluted in an egg yolk±Tris±glycerol extender. The semen was frozen
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Table 1 pH, osmolarity and viscosity of migration medium 1, 2 and 3 with 0.27, 0.68 and 1.35% MC, respectively, determined at four rotation speeds Medium
1 2 3
Concentration of MC (%)
0.27 0.68 1.35
pH
7.1 7.1 7.1
Osmolarity
295 289 287
Viscosity (mPa s) 64 rpm
128 rpm
256 rpm
512 rpm
6.8 16.5 65.5
5.4 13.4 53.0
4.5 11.6 53.0
4.9 9.8 31.4
in 0.25 ml straws and maintained in liquid nitrogen at 196 8C until use. Frozen semen of the same batch was used throughout the experiment. To determine the repeatability of the sperm migration assay, frozen semen of six bulls was used. The frozen-thawed semen used for the determination of the NRRs originated from the same batch as the semen used for the sperm migration test. 2.1.3. Sperm migration test The in vitro migration apparatus consisted of nonheparinised round microhaematocrite capillaries (Hirschmann1, Eberstadt, Germany) which were ®xed in plastic petridishes to facilitate handling. In combination with inverted microscopy, round capillaries are as suitable as specially designed ¯at tubes [5]. The length of the capillaries was 7.5 cm and they had an internal diameter of 1.5 mm. They were ®lled with the selected migration medium and closed at one end with putty. At the other end, 10 ml of space was left in which the sperm suspension was injected. The capillaries were prewarmed in a humidi®ed chamber at 37 8C. Only capillaries free of air bubbles were used. At time zero (t0), 10 ml of sperm suspension was injected in the open end of the capillary. The migration distance was determined at different time intervals after injection by means of an inverted light microscope (Olympus1 IX70-S8F, Japan) equipped with a heating stage at 37 8C at magni®cation of 200 (Fig. 1). The capillaries were examined microscopically from the end where the sperm was injected to the most distant point containing motile spermatozoa. Since we were interested in spermatozoa with a good migration capacity, the `migration distance' for the vanguard one and ®ve spermatozoa (including the vanguard one) was measured (millimetres). The migration distance of the vanguard spermatozoa was measured from the place of injection to the farthest point of migration. 2.2. Experimental design and statistical analysis 2.2.1. Experiment 1 In¯uence of MC concentration on the sperm migration distance. In this experiment, for each concentration of MC (0.27, 0.68 and 1.35% MC) six replicates per bull were used. The test was performed with frozen-thawed semen of six bulls. After thawing the semen, 1 ml of Hepes-TALP was added and the sperm suspension was centrifuged for 5 min at 514 g. The supernatant was removed after centrifugation and the spermatozoa were diluted to obtain a ®nal concentration of 120 106 spermatozoa/ml. Sperm quality was determined before injection into the capillary tubes for the sperm migration test. The migration
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Fig. 1. Determination of sperm migration distance in vitro by means round microhaematocrite capillaries, which were ®xed in plastic petridishes to facilitate handling, in combination with inverted microscopy.
distance was determined for the one and ®ve vanguard motile spermatozoa at 15 and 30 min after injection of the spermatozoa into the capillary tubes and incubation in the humidi®ed chamber at 37 8C. The comparison of the different media and different spermatozoa concentrations in relation to the sperm migration distance was done by using ANOVA (SPSS 10, SPSS Inc., IL 60611, USA, 1999). 2.2.2. Experiment 2 Repeatability of the sperm migration test. Semen from six bulls was used to measure migration in 1.35% MC. Per bull, six replicates of the sperm migration test were performed. In each capillary 120 106 spermatozoa/ml of spermatozoa were injected and the migration distance of the one and ®ve vanguard motile spermatozoa was determined at 15 (t1), 30 (t2), 45 (t3) and 60 min (t4) after incubation in the humidi®ed chamber at 37 8C. The repeatability of the sperm migration test was determined by evaluating the coef®cient of variation (CV) for the one and ®ve vanguard motile spermatozoa. 2.2.3. Experiment 3 In¯uence of sperm concentration on the sperm migration distance. Semen from two bulls was used to perform the sperm migration assay in a medium with 1.35% MC. The frozenthawed semen was centrifuged for 5 min at 514 g and concentrated to 100 106 and 150 106 spermatozoa/ml. For each concentration, six replicates per bull were tested. In contrast to the previous experiments, the migration distance was not determined by assessing the migration point of the one and ®ve vanguard spermatozoa but was determined by the most distant point containing ®ve motile spermatozoa per microscopic ®eld at t0 (moment of injection), t1 (15 min) and t2 (30 min) after injection of the spermatozoa. The in¯uence of the two sperm concentrations (100 106 and 150 106 spermatozoa/ml) on the sperm migration distance was determined by means of the independent sample t-test (SPSS 10, SPSS Inc., IL 60611, USA, 1999).
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2.2.4. Experiment 4 Relation between four sperm quality parameters and their predictive value for NRRs. Frozen-thawed semen from ®ve bulls with NRRs ranging from 52.8 to 66.7% was used to determine progressive motility, membrane integrity, sperm concentration and the sperm migration distance. The spermatozoa used for the sperm migration assay were of the same batch as those used for the determination of in vivo fertility. The NRRs were determined after 219 16 (mean S:D:) ®rst inseminations in heifers and cows. The inseminations were performed 12±18 h after the ®rst estrous signs observed by the farmer. The NRRs were not known by the investigators at the time the sperm migration experiments were performed. Prior to freezing, the semen from the ®ve bulls exhibited minimum values of 80% progressive motility, 80% membrane intact spermatozoa and a concentration of at least 0:75 109 spermatozoa/ml. After thawing, the percentage of progressive motile and membrane intact spermatozoa and sperm concentration ranged from 25±50 to 62±86% and 6 106 to 14 106 spermatozoa/ml, respectively, for the ®ve bulls. The progressive motility of the bull semen was evaluated subjectively at 37 8C. Eosin± nigrosin staining was used for the assessment of membrane integrity. The sperm concentration was determined by means of a BuÈrker counting chamber. These three basic parameters were evaluated by means of light microscopy. The sperm migration capacity was determined in the Hepes-TALP medium containing 1.35% MC. Six replicates of the sperm migration assay were performed per bull. The migration distance of the ®ve vanguard spermatozoa was measured at 15 min (t1) and 30 min (t2) after injecting 120 106 spermatozoa/ml. The correlation between the sperm migration distance at t2 and the three other sperm quality parameters, as well as the correlation between the NRRs of the ®ve bulls, and the progressive motility, membrane integrity and sperm migration distance at t2, was determined by calculating the correlation coef®cient (r). Linear regression was used to evaluate the predictive properties of the four sperm quality parameters on in vivo fertility (NRR), as well as to evaluate the relation between the sperm migration distance and the three other sperm quality parameters (SPSS 10, SPSS Inc., IL 60611, USA, 1999). 3. Results 3.1. Experiment 1: In¯uence of MC concentration on the sperm migration distance The migration distance of the one and ®ve vanguard spermatozoa after 15 min of incubation was signi®cantly shorter in the medium with the highest viscosity than in the two other media. After 30 min of incubation, the migration distance for the one and ®ve vanguard motile spermatozoa was signi®cantly different for the three media (P < 0:05) (Table 2). The most viscous medium (1.35% MC) yielded the lowest mean sperm migration distance. 3.2. Experiment 2: Repeatability of the sperm migration test From Experiment 2, only medium 3 was used for the sperm migration assay. The repeatability of the sperm migration assay in the medium with 1.35% MC for all six bulls is
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Table 2 Migration distance in mm (mean S:E:M:) for six replicates per bull (n 6) of the vanguard one and five motile spermatozoa after 15 and 30 min incubation in migration medium with three different concentrations of MC Medium
Migration distance at 15 min Vanguard one
0.27% MC 0.68% MC 1.35% MC
Migration distance at 30 min
Vanguard five
a
a
30.9 0.23 28.6 0.23a 18.3 0.23b
26.8 0.19 23.6 0.19a 16.0 0.19b
Vanguard one c
44.9 0.27 38.3 0.27d 25.7 0.27e
Vanguard five 40.4 0.26c 32.8 0.26d 21.4 0.26e
Values with different superscripts (a, b, c, d, e) in the same column are signi®cantly different (P < 0:05).
presented in Table 3. The repeatability of the sperm migration assay increased with increasing incubation time and was higher for the ®ve vanguard spermatozoa than for the one vanguard spermatozoon. It was noticed that the number of motile spermatozoa decreased substantially after >30 min incubation at 37 8C. Therefore, the sperm migration at a later point of time was not considered reliable despite the low CV. From Table 3, it is clear that the highest repeatability at t2 is obtained for the ®ve vanguard spermatozoa (CV 10:7 3:7). 3.3. Experiment 3: In¯uence of sperm concentration on the sperm migration distance Experiment 3, although the ®ve vanguard spermatozoa at t1 and t2 of incubation showed a tendency to migrate more at a concentration of 150 106 spermatozoa/ml than at a concentration of 100 106 spermatozoa/ml, this difference was only signi®cant at t1 (P 0:89 at t0, P 0:02 at t1, P 0:06 at t2) (Fig. 2). 3.4. Experiment 4: Relation between four different sperm quality parameters and their predictive value for NRRs Experiment 4 as is shown in Table 4, sperm concentration (P < 0:01) and membrane integrity (P 0:05) were signi®cantly related to the sperm migration distance at t2. Moreover, the sperm concentration could explain 64% of the variation in migration Table 3 Repeatability of the sperm migration assay determined by the CV for the vanguard one and five motile spermatozoa in migration medium with 1.35% MC Time of incubation (min) 15 30 45 60
(t1) (t2) (t3) (t4)
Average
Coefficient of variation Vanguard one spermatozoon
Vanguard five spermatozoa
11.4 11.0 10.0 8.1
11.1 10.7 7.5 5.4
10.0
8.5
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Fig. 2. In¯uence of sperm concentration on sperm migration distance at 0, 15 and 30 min after injection of spermatozoa in the capillaries. Table 4 Relation between progressive motility, membrane integrity, sperm concentration and sperm migration distance of bovine spermatozoa was tested at 30 min of incubation in a migration medium of 1.35% methyl cellulose Sperm migration distance at t2 b0 Progressive motility Membrane integrity Concentration
36.48 26.27 22.00
b1 0.02 0.14 0.15
P
r
0.81 0.05 <0.01
0.05 0.36 0.64
Linear regression was used to determine the regression coef®cients (b0 and b1) and the correlation coef®cient (r).
distance. The correlation between progressive motility and the sperm migration distance was very low (r 0:05). As is presented in Table 5, it is obvious that the progressive motility was the sperm quality parameter which is most correlated (r 0:66, P < 0:01) to the NRRs of the ®ve bulls. The sperm migration distance at t1 and t2 was also signi®cantly related to the NRRs, albeit in a negative way (b1 0:50 and 0.43, respectively). The correlation between membrane integrity and NRRs was negligible (r 0:05). Table 5 Use of sperm quality parameters: progressive motility, membrane integrity, migration distance after 15 min (t1) and 30 min (t2) incubation in a medium of 1.35% methyl cellulose to predict NRR NRR b0 Progressive motility Membrane integrity Migration distance at t1 Migration distance at t2
49.32 58.19 74.75 74.69
b1 0.32 0.02 0.50 0.43
P
r
<0.01 0.81 <0.01 0.02
0.66 0.05 0.52 0.43
Linear regression was used to determine the regression coef®cients (b0 and b1) and the correlation coef®cient (r).
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4. Discussion The capacity for sperm migration through cervical mucus depends on the structural± mechanical properties of the mucus microstructure, the viscosity of the mucus plasma and the size and shape of the sperm head and ¯agellum [3]. In this study, we used a synthetic medium based upon MC instead of cervical mucus to obtain a sperm migration medium that could be tested for its suitability to predict in vivo bull fertility. Cervical mucus of estrous cattle has a pH of about 7, a viscosity of 23.2 mm H2O, and a spinnbarkeit of 72 mm. Spermatozoa can migrate in cervical mucus on average a distance of 13 mm for the ®rst 10 min [25]. The migration medium with the highest concentration of MC (1.35% MC) had a pH of 7.1, and spermatozoa migrated a distance of 16 mm within 15 min, which is very similar to the values obtained in bovine cervical mucus. However, the spinnbarkeit of our MC medium was only 7 mm, and unlike polyacrylamide or cervical mucus, it did not have the appearance of a gel. The 1.35% MC medium had a viscosity of 65.5 mPa s but because of the difference in units, it is dif®cult to compare the viscosity of the MC medium with that of cervical mucus. From Experiment 1, it is obvious that sperm movement was depressed when the viscosity of the sperm migration medium increased. Since the sperm migration capacity in the 1.35% MC medium and cervical mucus were relatively similar, and the length of the capillary tubes was not a limiting factor for sperm migration capacity in the 1.35% MC medium, this medium was used for further experiments. Methyl cellulose is an unstructured and unreactive buffer that interferes with sperm migration only because of its increased viscosity and was, therefore, perfectly suited for our purpose, which was to evaluate sperm migration through a synthetic medium with good repeatability. Sperm migration in a MC solution was quite repeatable as indicated by a CV of 10.7%. Sperm samples which were introduced into the capillary at two different concentrations showed a tendency to migrate further at a concentration of 150 106 spermatozoa/ml than at a concentration of 100 106 spermatozoa/ml, although this was only signi®cant at t1 (P 0:02). This tendency was con®rmed in Experiment 4, where concentration had a signi®cant positive correlation with the sperm migration distance. In a number of studies, sperm migration capacity has been linked with other sperm characteristics such as total motility, sperm morphology, progressive motility, acrosome integrity and sperm concentration [7,26]. In our study, only the correlations between sperm migration capacity and progressive motility, membrane integrity and sperm concentration were determined. In a recent study, Anilkumar et al. [27] found that sperm penetration in polyacrylamide gel and bovine cervical mucus was positively correlated with post-thaw motility (r 0:81; r 0:89: P < 0:01) and acrosome integrity (r 0:88; r 0:94: P < 0:01) of bovine spermatozoa. However, in contrast to this [27] and other studies [17,26], in our study no correlation was found between progressive motility and the sperm migration distance (r 0:05, P 0:81). In accordance with Galli et al. [26] and Goldstein et al. [17], we did found a positive correlation between the sperm migration distance and membrane integrity (r 0:36, P 0:05) and concentration (r 0:64, P < 0:01). Although sperm migration capacity has been linked with different sperm quality parameters, it has seldom been linked with in vivo fertility in ruminants [28]. In contrast to studies in humans [6,17], we were not able to establish a positive correlation between the sperm migration distance in a migration medium with 1.35% MC and the NRRs of ®ve
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proven sires. The sperm migration distance at t1 and t2 was even negatively related (b1 0:50 and 0.43, respectively; P < 0:01 and 0.02, respectively) to the NRRs. In contrast to this, bull fertility and the sperm migration distance were linked in a positive way in a previous study [28]. However, the experimental design in that study differed from ours in two ways: ®rst, the sperm migration assay was performed in cervical mucus, and second, fresh spermatozoa were used, whereas the NRRs were obtained after insemination with frozen-thawed spermatozoa. This means that different ejaculates were used for the sperm migration assay and for the determination of the NRRs, whereas in our study the frozenthawed semen used for the sperm migration test and for the determination of the NRRs originated from the same batch. Apart from the experimental design, there are several explanations that may explain the disparity in results between different bovine studies and between bovine and human experiments. First, it could be due to differences in characteristics between the media used. Despite the fact that the MC medium yielded repeatable results it might not be as effective as hyaluronate, cervical mucus or polyacrylamide gel for sperm migration tests. Hyaluronate and cervical mucus possess a particular alignment of mucin chains resulting in a higher viscosity and in sperm movement parallel to the axis of the tube into mucus and hyaluronate solution channels [9]. In contrast to cervical mucus, the sperm penetrability in synthetic media such as MC and hyaluronate is not impaired by the presence of anti-sperm antibodies [9]. Polyacrylamide has proved to be more effective than MC when it comes to favouring migration of hyperactivated spermatozoa, at least in hamsters [24]. Whether this property of the medium is really important for studying bovine sperm migration remains to be determined, since it is known that bull spermatozoa are not hyperactivated at ejaculation, although they still have to penetrate the cervical mucus at this point of time [24]. Despite the fact that frozen-thawed bull spermatozoa are prone to premature capacitation [29], they do not hyperactivate as readily as hamster or mouse spermatozoa [24,30], and so it might be dif®cult to ®nd a relation between sperm migration and fertility in bulls, even if a more suitable medium such as polyacrylamide gel was used. Second, in contrast to humans, in cattle the difference in sperm quality between bulls is a lot smaller and the number of individuals with very poor sperm quality is very low. For the evaluation of the sperm migration test in humans, spermatozoa of fertile men (donors of an AI program) were compared to spermatozoa of patients attending an infertility laboratory [6,31]. Whereas in cattle the sperm migration test was performed with spermatozoa of bulls used for arti®cial insemination. These bulls have a high fertility resulting in a narrow range of NRRs, and therefore, the number of individuals with poor sperm quality in cattle is low [5]. In the only other comparable study of cattle, a relation was found between fertility and sperm migration, but interestingly, in this study one bull with 50% NRR and one with 14% NRR were compared with 11 bulls with NRRs ranging from 60 to 70% [28]. If the two bulls with the lowest NRRs were not included in the experiment, no relation could be found between NRR and the sperm migration distance. Even in the most carefully designed experiment in terms of examining the same ejaculate for sperm migration as for NRRs, sperm migration would probably only discriminate between bulls with extremely varying NRRs. Finally, the absence of a relation between sperm migration capacity and bull fertility could be explained by the fact that in dairy cattle, NRRs are determined after arti®cial insemination. During insemination the cervical barrier that spermatozoa have to overcome
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after natural mating is bypassed and the semen is deposited in the uterine body or in the uterine horns. It would be more logical to correlate sperm migration tests with fertility data obtained after natural mating, because then the spermatozoa have to migrate through the mucus before they reach the site of fertilization. It has been shown that sperm migration through cervical mucus in vitro could be indicative of the capacity of spermatozoa to populate the oviduct of goats [32]. After semen deposition at the external cervical os, spermatozoa from males with a high migration ef®ciency populated the oviduct more effectively than that from males with low migration ef®ciency, however differences were not signi®cant. However, this relation was not re¯ected in in vitro fertilization rates [32]. This may imply that capillary sperm migration tests may have value in predicting fertility under in vivo conditions, because the test is able to select superior sperm samples. In conclusion, we have shown that sperm migration through MC, albeit a repeatable test, could not be linked with in vivo bull fertility. Sperm migration through a viscous medium is only a very limited aspect of sperm behaviour that has to be performed in preparation to fertilize the oocyte. To obtain more precise prediction of in vivo fertility, it is imperative to combine different in vitro test systems as had been proposed by several authors [33,34]. Acknowledgements This research was supported by the Belgian Ministry of Agriculture, Grant no. S5867 and by the research fund of Ghent University, Grant no. 011B8698. References [1] Suarez SS, Drost M, Redfern K, Gottlieb W. Sperm motility in the oviduct. In: Bavister BD, Cummins J, Roland ERS, editors. Fertilization in mammals. Norwell MA: Serono Symposia, 1990. p. 111±24. [2] Hunter RHF. Human fertilization in vivo, with special reference to progression, storage and release of competent spermatozoa. Hum Reprod 1987;2:329±32. [3] Katz DF, Drobnis EZ, Overstreet JW. Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamet Res 1989;22:443±69. [4] Reichmann J, Insler V, Serr DV. A modi®ed in vitro spermatozoal penetration test. Int J Fertil 1973;18:241±5. [5] Kummerfeld HL, Lorton SP, Foote RH. Relationship of spermatozoal migration in cervical mucus to bovine fertility. J Androl 1981;2:103±7. [6] Alexander NJ. Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril 1981;36:201±8. [7] Hull MGR, McLeod FN, Joyce DN, Ray BD, McDermott A. Human in vitro fertilization, in vivo sperm penetration of cervical mucus and unexplained infertility. Lancet 1984;2:245±8. [8] Lee WI, Gaddum-Rosse P, Blandau RJ. Sperm penetration into cervical mucus in vitro. Part III. Effect of freezing on estrous bovine cervical mucus. Fertil Steril 1981;36:209±13. [9] Tang S, Garrett C, Gordon Baker HW. Comparison of human cervical mucus and arti®cial sperm penetration media. Hum Reprod 1999;14:2812±7. [10] Aitken RJ, Bowie H, Buckingham D, Harkiss D, Richardson DW, West KM. Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence. J Androl 1992;13:44±54. [11] Eggert-Kruse W, Gerhard I, Tilgen W, Runnebaum B. The use of hens egg white as a substitute for human cervical mucus in assessing human infertility. Int J Androl 1990;13:258±66.
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