Fluorescent labelling of boar spermatozoa for quantitative studies on competitive sperm–oviduct binding
Heiko H. W. Henning![https://orcid.org/0000-0003-4064-7792](/media/client/orcid_16x16.png)
A Unit for Reproductive Medicine, Clinic for Pigs and Small Ruminants, University of Veterinary Medicine, Bünteweg 15, 30559 Hannover, Germany.
B Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 112, 3584 CM Utrecht, Netherlands.
C Corresponding author. Email: dagmar.waberski@tiho-hannover.de
Reproduction, Fertility and Development 31(9) 1520-1532 https://doi.org/10.1071/RD19081
Submitted: 3 July 2018 Accepted: 8 April 2019 Published: 10 May 2019
Abstract
In vitro sperm–oviduct binding assays enable assessment of the capacity of spermatozoa to form a ‘reservoir’ in the oviduct. Competitive approaches, such as experimental set-ups that test multiple males or semen samples simultaneously on the same tissue explants, are desirable because they reduce the likelihood of bias when using material from different females. Therefore, we established a fluorescent labelling technique that allows tagging and storage of spermatozoa before competitive studies of sperm–oviduct binding in vitro. Fluorescent markers were tested for reliability and compatibility with parameters of boar spermatozoa viability. The addition of seminal plasma after density gradient centrifugation was essential to counteract centrifugation stress during the labelling procedure. It was demonstrated that sperm tagged with MitoTracker Green FM or MitoTracker Red FM can be successfully used in competitive sperm–oviduct binding studies. The assay was sensitive enough to indicate subtle effects of semen storage temperature on the ability of the spermatozoa to contribute to the female sperm reservoir.
Additional keywords: sperm competition, sperm reservoir.
References
Amann, R. P., Saacke, R. G., Barbato, G. F., and Waberski, D. (2018). Measuring male-to-male differences in fertility or effects of semen treatments. Annu. Rev. Anim. Biosci. 6, 255–286.| Measuring male-to-male differences in fertility or effects of semen treatments.Crossref | GoogleScholarGoogle Scholar | 29131658PubMed |
Braundmeier, A. G., Demers, J. M., Shanks, R. D., and Miller, D. J. (2004). The relationship of porcine sperm zona-binding ability to fertility. J. Anim. Sci. 82, 452–458.
| The relationship of porcine sperm zona-binding ability to fertility.Crossref | GoogleScholarGoogle Scholar | 14974543PubMed |
Chen, S., Einspanier, R., and Schoen, J. (2013). In vitro mimicking of estrous cycle stages in porcine oviduct epithelium cells: estradiol and progesterone regulate differentiation, gene expression, and cellular function. Biol. Reprod. 89, 54.
| In vitro mimicking of estrous cycle stages in porcine oviduct epithelium cells: estradiol and progesterone regulate differentiation, gene expression, and cellular function.Crossref | GoogleScholarGoogle Scholar | 23904510PubMed |
Daigneault, B. W., McNamara, K. A., Purdy, P. H., Krisher, R. L., Knox, R. V., Rodriguez-Zas, S. L., and Miller, D. J. (2015). Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment. Andrology 3, 558–568.
| Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment.Crossref | GoogleScholarGoogle Scholar | 25914302PubMed |
de Wagenaar, B., Berendsen, J. T. W., Bomer, J. G., Olthuis, W., van den Berg, A., and Segerink, L. I. (2015). Microfluidic single sperm entrapment and analysis. Lab Chip 15, 1294–1301.
| Microfluidic single sperm entrapment and analysis.Crossref | GoogleScholarGoogle Scholar | 25578490PubMed |
Druart, X., Cognié, J., Baril, G., Clément, F., Dacheux, J. L., and Gatti, J. L. (2009). In vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe genital tract. Reproduction 138, 45–53.
| In vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe genital tract.Crossref | GoogleScholarGoogle Scholar | 19423661PubMed |
Ferraz, M. A. M. M., Henning, H. H. W., Costa, P. F., Malda, J., Melchels, F. P., Wubbolts, R., Stout, T. A. E., Vos, P. L. A. M., and Gadella, B. M. (2017). Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation. Lab Chip 17, 905–916.
| Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.Crossref | GoogleScholarGoogle Scholar |
Fléchon, J.-E., and Hunter, R. H. F. (1981). Distribution of spermatozoa in the utero-tubal junction and isthmus of pigs, and their relationship with the luminal epithelium after mating: a scanning electron microscope study. Tissue Cell 13, 127–139.
| Distribution of spermatozoa in the utero-tubal junction and isthmus of pigs, and their relationship with the luminal epithelium after mating: a scanning electron microscope study.Crossref | GoogleScholarGoogle Scholar | 7194520PubMed |
Flesch, F. M., Voorhout, W. F., Colenbrander, B., van Golde, L. M., and Gadella, B. M. (1998). Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity. Biol. Reprod. 59, 1530–1539.
| Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity.Crossref | GoogleScholarGoogle Scholar | 9828202PubMed |
Garner, D. L. (2009). Hoechst 33342: the dye that enabled differentiation of living X- and Y-chromosome bearing mammalian sperm. Theriogenology 71, 11–21.
| Hoechst 33342: the dye that enabled differentiation of living X- and Y-chromosome bearing mammalian sperm.Crossref | GoogleScholarGoogle Scholar | 18952273PubMed |
Garrels, W., Holler, S., Cleve, N., Niemann, H., Ivics, Z., and Kues, W. A. (2012). Assessment of fecundity and germ line transmission in two transgenic pig lines produced by sleeping beauty transposition. Genes (Basel) 3, 615–633.
| Assessment of fecundity and germ line transmission in two transgenic pig lines produced by sleeping beauty transposition.Crossref | GoogleScholarGoogle Scholar | 24705079PubMed |
Gatica, L. V., Guidobaldi, H. A., Montesinos, M. M., Teves, M. E., Moreno, A. I., Unates, D. R., Molina, R. I., and Giojalas, L. C. (2013). Picomolar gradients of progesterone select functional human sperm even in subfertile samples. Mol. Hum. Reprod. 19, 559–569.
| Picomolar gradients of progesterone select functional human sperm even in subfertile samples.Crossref | GoogleScholarGoogle Scholar | 23729411PubMed |
Gualtieri, R., and Talevi, R. (2003). Selection of highly fertilization-competent bovine spermatozoa through adhesion to the Fallopian tube epithelium in vitro. Reproduction 125, 251–258.
| Selection of highly fertilization-competent bovine spermatozoa through adhesion to the Fallopian tube epithelium in vitro.Crossref | GoogleScholarGoogle Scholar | 12578539PubMed |
Gualtieri, R., Mollo, V., Duma, G., and Talevi, R. (2009). Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation. Reproduction 138, 33–43.
| Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation.Crossref | GoogleScholarGoogle Scholar | 19439561PubMed |
Gualtieri, R., Mollo, V., Braun, S., Barbato, V., Fiorentino, I., and Talevi, R. (2013). Bovine oviductal monolayers cultured under three-dimension conditions secrete factors able to release spermatozoa adhering to the tubal reservoir in vitro. Theriogenology 79, 429–435.
| Bovine oviductal monolayers cultured under three-dimension conditions secrete factors able to release spermatozoa adhering to the tubal reservoir in vitro.Crossref | GoogleScholarGoogle Scholar | 23168352PubMed |
Heifetz, Y., and Rivlin, P. K. (2010). Beyond the mouse model: using Drosophila as a model for sperm interaction with the female reproductive tract. Theriogenology 73, 723–739.
| Beyond the mouse model: using Drosophila as a model for sperm interaction with the female reproductive tract.Crossref | GoogleScholarGoogle Scholar | 20015541PubMed |
Henning, H., Petrunkina, A. M., Harrison, R. A., and Waberski, D. (2012). Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis. Cytometry A 81A, 576–587.
| Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis.Crossref | GoogleScholarGoogle Scholar |
Henning, H., Ngo, T. T., and Waberski, D. (2015). Centrifugation stress reduces the responsiveness of spermatozoa to a capacitation stimulus in in vitro-aged semen. Andrology 3, 834–842.
| Centrifugation stress reduces the responsiveness of spermatozoa to a capacitation stimulus in in vitro-aged semen.Crossref | GoogleScholarGoogle Scholar | 26226856PubMed |
Hoffmann, O. I., Kerekes, A., Liptak, N., Hiripi, L., Bodo, S., Szaloki, G., Klein, S., Ivics, Z., Kues, W. A., and Bosze, Z. (2016). Transposon-based reporter marking provides functional evidence for intercellular bridges in the male germline of rabbits. PLoS One 11, e0154489.
| Transposon-based reporter marking provides functional evidence for intercellular bridges in the male germline of rabbits.Crossref | GoogleScholarGoogle Scholar | 27148973PubMed |
Holt, W. V., and Fazeli, A. (2016). Sperm storage in the female reproductive tract. Annu. Rev. Anim. Biosci. 4, 291–310.
| Sperm storage in the female reproductive tract.Crossref | GoogleScholarGoogle Scholar | 26526545PubMed |
Hossain, M. S., Johannisson, A., Wallgren, M., Nagy, S., Siqueira, A. P., and Rodriguez-Martinez, H. (2011). Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J. Androl. 13, 406–419.
| Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art.Crossref | GoogleScholarGoogle Scholar | 21478895PubMed |
Hunter, R. H. E. (1981). Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation. J. Reprod. Fertil. 63, 109–117.
| Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation.Crossref | GoogleScholarGoogle Scholar |
Hunter, R. H., Flechon, B., and Flechon, J. E. (1991). Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study. Tissue Cell 23, 641–656.
| Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study.Crossref | GoogleScholarGoogle Scholar | 1776154PubMed |
Johnson, I. D., and Spence, M. T. Z. (2010). ‘Molecular Probe Handbook: A Guide to Fluorescent Probes and Labeling Technologies.’ (Molecular Probes: Eugene, OR.)
Johnson, L. A., Weitze, K. F., Fiser, P., and Maxwell, W. M. (2000). Storage of boar semen. Anim. Reprod. Sci. 62, 143–172.
| Storage of boar semen.Crossref | GoogleScholarGoogle Scholar | 10924823PubMed |
Khalil, A. A., Petrunkina, A. M., Sahin, E., Waberski, D., and Töpfer-Petersen, E. (2006). Enhanced binding of sperm with superior volume regulation to oviductal epithelium. J. Androl. 27, 754–765.
| Enhanced binding of sperm with superior volume regulation to oviductal epithelium.Crossref | GoogleScholarGoogle Scholar | 16809276PubMed |
Leemans, B., Gadella, B. M., Stout, T. A., Heras, S., Smits, K., Ferrer-Buitrago, M., Claes, E., Heindryckx, B., De Vos, W. H., Nelis, H., Hoogewijs, M., and Van Soom, A. (2015). Procaine induces cytokinesis in horse oocytes via a pH-dependent mechanism. Biol. Reprod. 93, 23.
| Procaine induces cytokinesis in horse oocytes via a pH-dependent mechanism.Crossref | GoogleScholarGoogle Scholar | 26085521PubMed |
Liu, D. Y., and Baker, H. W. (1990). Inducing the human acrosome reaction with a calcium ionophore A23187 decreases sperm-zona pellucida binding with oocytes that failed to fertilize in vitro. J. Reprod. Fertil. 89, 127–134.
| Inducing the human acrosome reaction with a calcium ionophore A23187 decreases sperm-zona pellucida binding with oocytes that failed to fertilize in vitro.Crossref | GoogleScholarGoogle Scholar | 2115581PubMed |
Lüpold, S., Manier, M. K., Berben, K. S., Smith, K. J., Daley, B. D., Buckley, S. H., Belote, J. M., and Pitnick, S. (2012). How multivariate ejaculate traits determine competitive fertilization success in Drosophila melanogaster. Curr. Biol. 22, 1667–1672.
| How multivariate ejaculate traits determine competitive fertilization success in Drosophila melanogaster.Crossref | GoogleScholarGoogle Scholar | 22840512PubMed |
Lymbery, R. A., Kennington, W. J., and Evans, J. P. (2016). Fluorescent sperm offer a method for tracking the real-time success of ejaculates when they compete to fertilise eggs. Sci. Rep. 6, 22689.
| Fluorescent sperm offer a method for tracking the real-time success of ejaculates when they compete to fertilise eggs.Crossref | GoogleScholarGoogle Scholar | 26941059PubMed |
Maxwell, W. M., and Johnson, L. A. (1999). Physiology of spermatozoa at high dilution rates: the influence of seminal plasma. Theriogenology 52, 1353–1362.
| Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.Crossref | GoogleScholarGoogle Scholar | 10735081PubMed |
Mellish, K. S., and Baker, R. D. (1970). Marking boar spermatozoa with fluorochromes for evaluating spermatozoan transport within gilts. J. Anim. Sci. 31, 917–922.
| Marking boar spermatozoa with fluorochromes for evaluating spermatozoan transport within gilts.Crossref | GoogleScholarGoogle Scholar | 5481267PubMed |
Mellish, K. S., Baker, R. D., and Moxley, J. E. (1968). Transport of F.I.T.C.-conjugated and unmarked sperm within gilts. J. Anim. Sci. 27, 1787.
Miller, D. J., Demers, J. M., Braundmeier, A. G., and Behrens, M. L. (1998). The use of two fluorescent dyes to identify sperm in a competitive binding assay to oocytes. J. Androl. 19, 650–656.
| 9876016PubMed |
Muro, Y., Hasuwa, H., Isotani, A., Miyata, H., Yamagata, K., Ikawa, M., Yanagimachi, R., and Okabe, M. (2016). Behavior of mouse spermatozoa in the female reproductive tract from soon after mating to the beginning of fertilization. Biol. Reprod. 94, 80.
| Behavior of mouse spermatozoa in the female reproductive tract from soon after mating to the beginning of fertilization.Crossref | GoogleScholarGoogle Scholar | 26962112PubMed |
Niu, Y., Greube, A., Ji, W., and Jewgenow, K. (2006). The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm. Theriogenology 66, 989–995.
| The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm.Crossref | GoogleScholarGoogle Scholar | 16620930PubMed |
Nguyen, Q. T., Wallner, U., Schmicke, M., Waberski, D., and Henning, H. (2016). Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction. Biol. Open 5, 1743–1751.
| Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.Crossref | GoogleScholarGoogle Scholar | 27612509PubMed |
Overstreet, J. W., and Cooper, G. W. (1978). Sperm transport in the reproductive tract of the female rabbit: II. The sustained phase of transport. Biol. Reprod. 19, 115–132.
| Sperm transport in the reproductive tract of the female rabbit: II. The sustained phase of transport.Crossref | GoogleScholarGoogle Scholar | 567499PubMed |
Parrilla, I., Vázquez, J. M., Cuello, C., Gil, M. A., Roca, J., Di Berardino, D., and Martínez, E. A. (2004). Hoechst 33342 stain and u.v. laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa. Reproduction 128, 615–621.
| Hoechst 33342 stain and u.v. laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa.Crossref | GoogleScholarGoogle Scholar | 15509707PubMed |
Parrish, J. J., and Foote, R. H. (1985). Fertility differences among male rabbits determined by heterospermic insemination of fluorochrome-labeled spermatozoa. Biol. Reprod. 33, 940–949.
| Fertility differences among male rabbits determined by heterospermic insemination of fluorochrome-labeled spermatozoa.Crossref | GoogleScholarGoogle Scholar | 3936554PubMed |
Pérez-Cerezales, S., Laguna-Barraza, R., de Castro, A. C., Sánchez-Calabuig, M. J., Cano-Oliva, E., de Castro-Pita, F. J., Montoro-Buils, L., Pericuesta, E., Fernández-González, R., and Gutiérrez-Adán, A. (2018). Sperm selection by thermotaxis improves ICSI outcome in mice. Sci. Rep. 8, 2902.
| Sperm selection by thermotaxis improves ICSI outcome in mice.Crossref | GoogleScholarGoogle Scholar | 29440764PubMed |
Petrunkina, A. M., Gehlhaar, R., Drommer, W., Waberski, D., and Töpfer-Petersen, E. (2001). Selective sperm binding to pig oviductal epithelium in vitro. Reproduction 121, 889–896.
| Selective sperm binding to pig oviductal epithelium in vitro.Crossref | GoogleScholarGoogle Scholar | 11373175PubMed |
Presley, A. D., Fuller, K. M., and Arriaga, E. A. (2003). MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 793, 141–150.
| MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.Crossref | GoogleScholarGoogle Scholar | 12880861PubMed |
Rath, D., and Johnson, L. A. (2008). Application and commercialization of flow cytometrically sex-sorted semen. Reprod. Domest. Anim. 43, 338–346.
| Application and commercialization of flow cytometrically sex-sorted semen.Crossref | GoogleScholarGoogle Scholar | 18638144PubMed |
Richardson, L., Hanrahan, J. P., O’Hara, L., Donovan, A., Fair, S., O’Sullivan, M., Carrington, S. D., Lonergan, P., and Evans, A. C. (2011). Ewe breed differences in fertility after cervical AI with frozen–thawed semen and associated differences in sperm penetration and physicochemical properties of cervical mucus. Anim. Reprod. Sci. 129, 37–43.
| Ewe breed differences in fertility after cervical AI with frozen–thawed semen and associated differences in sperm penetration and physicochemical properties of cervical mucus.Crossref | GoogleScholarGoogle Scholar | 22115522PubMed |
Rickard, J. P., Pini, T., Soleilhavoup, C., Cognie, J., Bathgate, R., Lynch, G. W., Evans, G., Maxwell, W. M., Druart, X., and de Graaf, S. P. (2014). Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa. Reproduction 148, 469–478.
| Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa.Crossref | GoogleScholarGoogle Scholar | 25118301PubMed |
Rottmayer, R., Ulbrich, S. E., Kölle, S., Prelle, K., Neumueller, C., Sinowatz, F., Meyer, H. H., Wolf, E., and Hiendleder, S. (2006). A bovine oviduct epithelial cell suspension culture system suitable for studying embryo–maternal interactions: morphological and functional characterization. Reproduction 132, 637–648.
| A bovine oviduct epithelial cell suspension culture system suitable for studying embryo–maternal interactions: morphological and functional characterization.Crossref | GoogleScholarGoogle Scholar | 17008475PubMed |
Schulze, M., Henning, H., Rüdiger, K., Wallner, U., and Waberski, D. (2013). Temperature management during semen processing: impact on boar sperm quality under laboratory and field conditions. Theriogenology 80, 990–998.
| Temperature management during semen processing: impact on boar sperm quality under laboratory and field conditions.Crossref | GoogleScholarGoogle Scholar | 23987989PubMed |
Suarez, S., Redfern, K., Raynor, P., Martin, F., and Phillips, D. M. (1991). Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir. Biol. Reprod. 44, 998–1004.
| Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir.Crossref | GoogleScholarGoogle Scholar | 1873399PubMed |
Sutovsky, P., Navara, C. S., and Schatten, G. (1996). Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization Biol. Reprod. 55, 1195–1205.
| Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilizationCrossref | GoogleScholarGoogle Scholar | 8949874PubMed |
Sutovsky, P., McCauley, T. C., Sutovsky, M., and Day, B. N. (2003). Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132. Biol. Reprod. 68, 1793–1800.
| Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132.Crossref | GoogleScholarGoogle Scholar | 12606393PubMed |
Thomas, P. G., and Ball, B. A. (1996). Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro. Mol. Reprod. Dev. 43, 55–61.
| Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro.Crossref | GoogleScholarGoogle Scholar | 8720113PubMed |
Vazquez, J. M., Martínez, E., Parrilla, I., Gil, M. A., Lucas, X., and Roca, J. (2002). Motility characteristics and fertilizing capacity of boar spermatozoa stained with Hoechst 33342. Reprod. Domest. Anim. 37, 369–374.
| Motility characteristics and fertilizing capacity of boar spermatozoa stained with Hoechst 33342.Crossref | GoogleScholarGoogle Scholar | 12464077PubMed |
Vincent, R., and Nadeau, D. (1984). Adjustment of osmolality of Percoll for the isopycnic separation of cells and cell organelles. Anal. Biochem. 141, 322–328.
| Adjustment of osmolality of Percoll for the isopycnic separation of cells and cell organelles.Crossref | GoogleScholarGoogle Scholar | 6093627PubMed |
Waberski, D., Dirksen, G., Weitze, K. F., Leiding, C., and Hahn, R. (1990). Field studies of the effect of sperm motility and morphology on the fertility of boars used for insemination. Tierarztl. Prax. 18, 591–594.
| 2080503PubMed |
Waberski, D., Magnus, F., Ardon, F., Petrunkina, A. M., Weitze, K. F., and Töpfer-Petersen, E. (2006). Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time. Reproduction 131, 311–318.
| Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time.Crossref | GoogleScholarGoogle Scholar | 16452724PubMed |
Winters, R. A., Hamilton, D. N., Bhatnagar, A. S., Fitzgerald, R., Bovin, N., and Miller, D. J. (2018). Porcine sperm binding to oviduct cells and glycans as supplements to traditional laboratory semen analysis. J. Anim. Sci. 96, 5265–5275.
| Porcine sperm binding to oviduct cells and glycans as supplements to traditional laboratory semen analysis.Crossref | GoogleScholarGoogle Scholar | 30252064PubMed |