Download PDF
Tierarztl Prax Ausg G Grosstiere Nutztiere 2013; 41(05): 297-303
DOI: 10.1055/s-0038-1623187
Original Article
Schattauer GmbH

Establishment of a practical sperm penetration test for bovine semen

Etablierung eines praktikablen In-vitro-Penetrationstests für bovine Samenzellen
A. C. Becher
1   Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig University Giessen, Giessen, Germany;
,
K. Failing
2   Unit of Biomathematics, Justus-Liebig-University, Giessen, Germany;
,
J. Kauffold
3   Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany
,
A. Wehrend
1   Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig University Giessen, Giessen, Germany;
› Author Affiliations
Further Information

Publication History

Received: 30 April 2013

Accepted after revision: 08 July 2013

Publication Date:
08 January 2018 (online)

    Permissions and Reprints

Summary

Objective: Several methods for evaluating semen quality have been developed in addition to traditional semen analysis. Most of these methods are very complex and require expensive technical equipment as well as special knowledge to be performed. The aim of this study was to establish an easy and cost-effective sperm penetration test for bovine semen using artificial media. Additionally a standard procedure for test performance should be figured out. Material and methods: In the first part of the study four different capillary tubes or pipettes were evaluated in order to determine the most appropriate one to use in the penetration test. In the second part of the study the tubes were filled with different compositions of polyacrylamide gel and subsequently incubated at 37.5 °C for 30 minutes. Sperm penetration was then evaluated using a phase contrast microscope, measuring the penetration distance of the first, the first motile and the five vanguard motile spermatozoa as well as determining the number of sperm cells cumulated per low power field (LPF) at three distinct distances in the tube. Results: Non-heparinised haematocrit capillary tubes were the most appropriate tubes for performance of sperm penetration test as 100% of used capillaries were evaluable after incubation and size of the capillaries allowed a simple handling and good visualisation of migrated spermatozoa. Best results were achieved with 1.9% polyacrylamide gel containing phosphate-buffered saline. Measuring the penetration distance of the vanguard motile sperm cell can be recommended for test evaluation. Conclusion: A sperm penetration test with 1.9% polyacrylamide using non-heparinised capillary tubes was established. Results showed high reproducibility, the procedure is simple to carry out and only requires a phase contrast microscope.

Zusammenfassung

Gegenstand und Ziel: Um den diagnostischen Wert der Spermauntersuchung zu erhöhen, wurden zusätzlich zu den Standardverfahren komplexere Analysemethoden entwickelt, die sich zumeist durch ho hen technischen Aufwand und hohe Kosten wenig praxistauglich zeigten. Ziel dieser Arbeit war, mithilfe künstlicher Medien einen einfachen und kostengünstigen In-vitro-Penetrationstest für bovine Samenzellen zu etablieren. Zudem sollte ein Standardverfahren zur Test auswertung erarbeitet werden. Material und Methoden: Im ersten Teil des Versuchs wurden vier verschiedene Kapillaren/Pailletten unterschiedlicher Größen dahingehend verglichen, inwieweit sie sich mit einem viskösen künstlichen Medium befüllen und mittels Phasenkontrastmikroskop durchmustern lassen. Im zweiten Versuchsteil wurde untersucht, wie Polyacrylamidgel als künstliches Medium in unterschiedlichen Konzentratio nen von bovinen Samenzellen penetriert wird. Hierzu wurde die Kapillare mit dem jeweiligen Medium befüllt, für 30 Minuten bei 37,5 °C in einem Samenreservoir inkubiert und mittels Phasenkontrastmikroskop durchgemustert. Notiert wurde die am weitesten zurückgelegte Pene trationsstrecke des ersten, des ersten lebenden sowie der ersten fünf lebenden Spermien. Ferner wurde die Anzahl der in einem Gesichtsfeld akkumulierten Spermatozoen an drei Distanzpunkten erfasst. Ergebnisse: Nichtheparinisierte Hämatokritkapillaren erwiesen sich am ge eignetsten für die Folgeversuche, da sie zu 100% auswertbar waren und ihre Abmessungen ein einfaches Handling sowie eine gute Sichtbarkeit migrierter Spermien erlaubten. Polyacrylamidgel mit einer Kon zentration von 1,9% unter Zugabe phosphatgepufferter Salzlösung eignete sich am besten für den Test. Die Erfassung der Penetrationsdistanz des vordersten lebenden Spermiums kann zur Auswertung empfohlen werden. Schlussfolgerung: Ein Spermien-Penetrationstest in 1,9%igem Polyacrylamidgel unter Verwendung nichtheparinisierter Hämatokritkapillaren konnte etabliert werden. Die Ergebnisse des Tests zeigten eine hohe Reproduzierbarkeit, die Testdurchführung ist einfach und erfordert lediglich ein Phasenkontrastmikroskop.

Key words

Penetration test - bovine semen - polyacrylamide gel

Schlüsselwörter

Penetrationstest - bovines Sperma - Polyacrylamidgel
 

  • References

  • 1 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 (01) 44-54.
    PubMedGoogle Scholar
  • 2 Alexander NJ. Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril 1981; 36 (02) 201-208.
    CrossrefPubMedGoogle Scholar
  • 3 Amit A, Bergman A, Yedwab G, David MP, Homonnai TZ, Paz G. Penetration of human ejaculated spermatozoa into human and bovine cervical mucus. II. Pattern and velocity of penetration. Int J Fertil 1982; 27 (03) 160-165.
    PubMedGoogle Scholar
  • 4 Anilkumar R, Devanathan TG, Pattabiraman SR, Edwin MJ. Correlation between the spermatozoal characteristics and sperm penetration distance in polyacrylamide gel and bovine cervical mucus. Theriogenology 2001; 55 (02) 685-691.
    CrossrefPubMedGoogle Scholar
  • 5 Bals-Pratsch M, Knuth UA, Nieschlag E. Does sperm penetration in bovine mucus provide additional information to the „classical“ spermiogram and motility characteristics?. Zentralbl Gynakol 1988; 110 (04) 224-231.
    PubMedGoogle Scholar
  • 6 Baruch AL, Yedwab G, David MP, Homonnai ZT, Paz GF. Establishment of a method for preservation of human cervical mucus for in vitro penetration tests. Gynecol Obstet Invest 1983; 15 (03) 163-170.
    CrossrefPubMedGoogle Scholar
  • 7 Bergman A, Amit A, David MP, Homonnai ZT, Paz GF. Penetration of human ejaculated spermatozoa into human and bovine cervical mucus. I. Correlation between penetration values. Fertil Steril 1981; 36 (03) 363-367.
    CrossrefPubMedGoogle Scholar
  • 8 Bergman A, Amit A, Yedwab G, David MP, Homonnai ZT, Paz GF. Filtering capacity of bovine cervical mucus towards abnormal forms of human-ejaculated spermatozoa. Int J Androl 1981; 4 (06) 675-684.
    CrossrefPubMedGoogle Scholar
  • 9 Correa JR, Pace MM, Zavos PM. Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology 1997; 48 (05) 721-731.
    CrossrefPubMedGoogle Scholar
  • 10 Cox JF, Zavala A, Saravia F, Rivas C, Gallardo P, Alfaro V. Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats. Theriogenology 2002; 58 (01) 9-18.
    CrossrefPubMedGoogle Scholar
  • 11 David MP, Amit A, Bergman A, Yedwab G, Paz GF, Homonnai ZT. Sperm penetration in vitro: correlations between parameters of sperm quality and the penetration capactiy. Fertil Steril 1979; 32 (06) 676-680.
    CrossrefPubMedGoogle Scholar
  • 12 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 (03) 540-549.
    CrossrefPubMedGoogle Scholar
  • 13 Gaddum-Rosse P, Blandau RJ, Lee WI. Sperm penetration into cervical mucus in vitro. II. Human spermatozoa in bovine mucus. Fertil Steril 1980; 33 (06) 644-648.
    CrossrefPubMedGoogle Scholar
  • 14 Galli A, Basetti M, Balduzzi D, Martignoni M, Bornaghi V, Maffii M. Frozen bovine semen quality and bovine cervical mucus penetration test. Theriogenology 1991; 35 (04) 837-844.
    CrossrefPubMedGoogle Scholar
  • 15 Gillan L, Kroetsch T, Maxwell WM, Evans G. Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls. Anim Reprod Sci 2008; 103 (3–4) 201-214.
    CrossrefPubMedGoogle Scholar
  • 16 Goericke-Pesch S, Failing K. Retrospective analysis of canine semen evaluations with special emphasis on the use of the hypoosmotic swelling (HOS) test and acrosomal evaluation using Spermac(®). Reprod Domest Anim 2013; 48 (02) 213-217.
    CrossrefPubMedGoogle Scholar
  • 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 (05) 668-674.
    CrossrefPubMedGoogle Scholar
  • 18 Hanson FW, Overstreet JW. The interaction of human spermatozoa with cervical mucus in vivo. Am J Obstet Gynecol 1981; 140 (02) 173-178.
    CrossrefPubMedGoogle Scholar
  • 19 Jeulin C, Soumah A, Jouannet P. Morphological factors influencing the penetration of human sperm into cervical mucus in vitro. Int J Androl 1985; 8 (03) 215-223.
    CrossrefPubMedGoogle Scholar
  • 20 Katz DF, Mills RN, Pritchett TR. The movement of human spermatozoa in cervical mucus. J Reprod Fertil 1978; 53 (02) 259-265.
    CrossrefPubMedGoogle Scholar
  • 21 Katz DF, Overstreet JW, Hanson FW. A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 1980; 33 (02) 179-186.
    CrossrefPubMedGoogle Scholar
  • 22 Kremer J. A simple sperm penetration test. Int J Fertil 1965; 10 (03) 209-215.
    PubMedGoogle Scholar
  • 23 Kummerfeld HL, Lorton SP, Foote RH. Relationship of spermatozoal migration in cervical mucus to bovine fertility. J Androl 1981 1981; 2 (02) 103-107.
    PubMedGoogle Scholar
  • 24 Lorton SP, Kummerfeld HL, Foote RH. Polyacrylamide as a substitute for cervical mucus in sperm migration tests. Fertil Steril 1981; 35 (02) 222-225.
    CrossrefPubMedGoogle Scholar
  • 25 Martinez-Rodriguez C, Alvarez M, Ordas L, Chamorro CA, Martinez-Pastor F, Anel L. et al. Evaluation of ram semen quality using polyacrylamide gel instead of cervical mucus in the sperm penetration test. Theriogenology 2012; 77 (08) 1575-1586.
    CrossrefPubMedGoogle Scholar
  • 26 Matousek J, Ríha J, Srsen V, Veselský L, Louda F. Penetration of cervical mucus and other body fluids by bull sperm in capillary tubes. Anim Reprod Sci 1989; 18 (1–3) 161-166.
    CrossrefPubMedGoogle Scholar
  • 27 Mills RN, Katz DF. A flat capillary tube system for assessment of sperm movement in cervical mucus. Fertil Steril 1978; 29 (01) 43-47.
    CrossrefPubMedGoogle Scholar
  • 28 Moghissi KS, Segal S, Meinhold D, Agronow SJ. In vitro sperm cervical mucus penetration: studies in human and bovine cervical mucus. Fertil Steril 1982; 37 (06) 823-827.
    CrossrefPubMedGoogle Scholar
  • 29 Mortimer D, Mortimer ST, Shu MA, Swart R. A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test. Hum Reprod 1990; 5 (07) 835-841.
    CrossrefPubMedGoogle Scholar
  • 30 Murase T, Braun JW. Impact of methodological factors on sperm penetration into cervical mucus in cattle. Theriogenology 1990; 34 (01) 73-80.
    CrossrefPubMedGoogle Scholar
  • 31 Murase T, Okuda K, Sato K. Assessment of bull fertility using a mucus pene tration test and a human chorionic gonadotrophin stimulation test. Theriogenology 1990; 34 (05) 801-812.
    CrossrefPubMedGoogle Scholar
  • 32 Ola B, Afnan M, Papaioannou S, Sharif K, Bjorndahl L, Coomarasamy A. Accuracy of sperm-cervical mucus penetration tests in evaluating sperm motility in semen: a systematic quantitative review. Hum Reprod 2003; 18 (05) 1037-1046.
    CrossrefPubMedGoogle Scholar
  • 33 Suarez SS, Dai X. Hyperactivation enhances mouse sperm capacity for penetrating viscoelastic media. Biol Reprod 1992; 46 (04) 686-691.
    CrossrefPubMedGoogle Scholar
  • 34 Suarez SS, Pacey AA. Sperm transport in the female reproductive tract. Hum Reprod Update 2006; 12 (01) 23-37.
    CrossrefPubMedGoogle Scholar
  • 35 Suttiyotin P, Thwaites CJ, Baillie ND. Relationships between the results of a modified sperm penetration test and a swim-up technique and the fertility of ram semen. Theriogenology 1992; 37 (04) 851-857.
    CrossrefPubMedGoogle Scholar
  • 36 Tas M, Bacinoglu S, Cirit Ü, Özgümüs S, Kasgöz H, Pabuccuoglu S. Estimation of the potential fertility based upon non-return rates of bulls: Using polyacrylamide gel instead of cervical mucus in the sperm penetration test. Theriogenology 2007; 68 (07) 981-987.
    CrossrefPubMedGoogle Scholar
  • 37 Töpfersen-Petersen E, Waberski D. Spermienreifung und Befruchtung. In: Veterinärmedizinische Andrologie. Busch W, Holzmann A. Hrsg. Stuttgart: Schattauer; 2001: 67-82.
    Google Scholar
  • 38 Verberckmoes S, Van Soom A, De Pauw I, Dewulf J, de Kruif A. Migration of bovine spermatozoa in a synthetic medium and its relation to in vivo bull fertility. Theriogenology 2002; 58 (05) 1027-1037.
    CrossrefPubMedGoogle Scholar
  • 39 von Stern K. Prognose der Tiefgefriereignung von Hengstsperma mit Hilfe von Funktionstests. Dissertation, Freie Universität Berlin, Tierklinik für Fortpflanzung. 2001
    PubMedGoogle Scholar
  • 40 WHO. Optional Procedures – Capillary Tube Test. In: WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th. edn. Geneva: WHO Press; 2010: 127-130.
    Google Scholar