Egg yolk-free cryopreservation of bull semen

Autoři: Muhammad Anzar aff001;  Kosala Rajapaksha aff001;  Lyle Boswall aff001
Působiště autorů: Agriculture and Agri-Food Canada, Saskatoon Research and Development Center, Saskatoon, Saskatchewan, Canada aff001;  Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223977


Egg yolk is a common ingredient of mammalian semen extender to protect sperm against initial cold shock. However, egg yolk has biosecurity risks. Our main objectives were to cryopreserve bull semen without egg yolk using exogenous cholesterol and to study the protective role of glycerol in egg yolk-free semen extender. Other objectives were to compare protein profiles and in vitro fertilization potential of bull sperm frozen with and without egg yolk. In first experiment, semen was either diluted in conventional tris-egg yolk glycerol (TEYG control) extender or first treated with cholesterol-cyclodextrin complex (CC, 2 mg/ml semen) followed by dilution in egg yolk-free tris-glycerol (TG) extender (collectively called as “CC+TG”) at 22°C or 4°C, and frozen. Post-thaw sperm motion characteristics were similar between CC+TG and TEYG control extenders, and temperature of glycerol addition. In second experiment, semen was frozen in CC+TG extender varying in glycerol concentration (7 to 0%; v/v). Post-thaw sperm quality decreased with the decline in glycerol concentration in TG extender, even higher concentration of CC complex (3 or 4 mg/ml semen) could not protect sperm in the absence of glycerol in TG extender. In third experiment, SDS electrophoresis of proteins from fresh sperm and sperm frozen in CC+TG, and TEYG control extenders was conducted. Protein profiles in fresh sperm and CC+TG frozen sperm were almost similar. Egg yolk proteins bound tightly with sperm plasma membrane. In fourth experiment, in vitro fertilization potentials of sperm frozen in TEYG control and CC+TG extenders were tested. Cleavage and blastocyst rates of semen frozen in CC+TG and TEYG control extenders were similar. In conclusion, cholesterol-cyclodextrin replaced egg yolk from the semen extender; glycerol remained essential for egg yolk-free sperm cryopreservation; and CC+TG extender did not modify sperm plasma membrane CC+TG whereas egg yolk extender changed the plasma membrane composition of bull sperm.

Klíčová slova:

Cell membranes – Cholesterol – Membrane proteins – Semen – Sperm – Glycerolization – Cryopreservation – Acrosomes


1. Phillips PH. The preservation of bulls semen. J Biol Chem. 1939;130: 415.

2. Pace MM, Graham EF. Components in egg yolk which protect bovine spermatozoa during freezing. J Anim Sci. 1974;39: 1144–1149. doi: 10.2527/jas1974.3961144x 4475048

3. Moussa M, Marinet V, Trimeche A, Tainturier D, Anton M. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. Theriogenology. 2002;57: 1695–1706. doi: 10.1016/s0093-691x(02)00682-9 12035979

4. Manjunath P, Chandonnet L, Leblond E, Desnoyers L. Major proteins of bovine seminal vesicles bind to spermatozoa. Biol Reprod. 1994;50: 27–37. doi: 10.1095/biolreprod50.1.27 8312447

5. Thérien I, Moreau R, Manjunath P. Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biol Reprod. 1999;61: 590–598. doi: 10.1095/biolreprod61.3.590 10456833

6. Bergeron A, Crete MH, Brindle Y, Manjunath P. Low-density lipoprotein fraction from hen's egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod. 2004;70: 708–717. doi: 10.1095/biolreprod.103.022996 14613896

7. Bergeron A, Manjunath P. New insights towards understanding the mechanisms of sperm protection by egg yolk and milk. Mol Reprod Dev. 2006;73: 1338–1344. doi: 10.1002/mrd.20565 16868925

8. de Ruigh L, Bosch JC, Brus MC, Landman B, Merton JS. Ways to improve the biosecurity of bovine semen. Reprod Dom Anim. 2006;41: 268–274.

9. van Wagtendonk-de Leeuw AM, Haring RM, Kaal-Lansbergen LM, den Daas JH. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract. Theriogenology. 2000;54: 57–67. doi: 10.1016/S0093-691X(00)00324-1 10990347

10. Thibier M, Guerin B. Hygienic aspects of storage and use of semen for artificial insemination. Anim Reprod Sci. 2000;62: 233–251. 10924827

11. CFIA (2002) Rules and regulations for imort and export of bilogical materials. In: Agency CFI, editor.

12. OIE (2016) Terrestrial Animal Health Code. World Organization of Animal Health.

13. Layek SS, Mohanty TK, Kumaresan A, Parks JE. Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders. Anim Reprod Sci. 2016;172: 1–9. doi: 10.1016/j.anireprosci.2016.04.013 27509873

14. Kumar P, Saini M, Kumar D, Balhara AK, Yadav SP, Singh P, et al. Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen. Anim Reprod Sci. 2015;159: 38–45. doi: 10.1016/j.anireprosci.2015.05.010 26065809

15. Ropke T, Oldenhof H, Leiding C, Sieme H, Bollwein H, Wolkers WF. Liposomes for cryopreservation of bovine sperm. Theriogenology. 2011;76: 1465–1472. doi: 10.1016/j.theriogenology.2011.06.015 21820724

16. Quinn PJ. Principles of membrane stability and phase behavior under extreme conditions. J Bioenerg Biomembr. 1989;21: 3–19. doi: 10.1007/bf00762209 2651426

17. Darin-Bennett A, White IG. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology. 1977;14: 466–470. doi: 10.1016/0011-2240(77)90008-6 560945

18. Davis BK. Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval. Proc Nat Acad Sci. 1981;78: 7560–7564. doi: 10.1073/pnas.78.12.7560 6950397

19. Moce E, Vicente JS. Rabbit sperm cryopreservation: a review. Anim Reprod Sci. 2009;110: 1–24. doi: 10.1016/j.anireprosci.2008.08.015 18805659

20. Parks JE, Graham JK. Effects of cryopreservation procedures on sperm membranes. Theriogenology. 1992;38: 209–222. doi: 10.1016/0093-691x(92)90231-f 16727131

21. Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon. J Androl. 2000;21: 1–7. 10670514

22. Choi YH, Toyoda Y. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol Reprod. 1998;59: 1328–1333. doi: 10.1095/biolreprod59.6.1328 9828175

23. Yu Y, Chipot C, Cai W, Shao X. Molecular dynamics study of the inclusion of cholesterol into cyclodextrins. J Phys Chem B. 2006;110: 6372–6378. doi: 10.1021/jp056751a 16553456

24. Purdy PH, Graham JK. Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology. 2004;48: 36–45. doi: 10.1016/j.cryobiol.2003.12.001 14969680

25. Moore AI, Squires EL, Graham JK. Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryobiology. 2005;51: 241–249. doi: 10.1016/j.cryobiol.2005.07.004 16122725

26. Galantino-Homer HL, Zeng WX, Megee SO, Dallmeyer M, Voelkl D, Dobrinski I. Effects of 2-hydroxypropyl-beta-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation. Mol Reprod Dev. 2006;73: 638–650. doi: 10.1002/mrd.20437 16450405

27. Blommaert D, Franck T, Donnay I, Lejeune JP, Detilleux J, Serteyn D. Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm. Cryobiology. 2016;72: 27–32. doi: 10.1016/j.cryobiol.2015.11.008 26687387

28. Lovelock JE. HET mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim Biophys Acta. 1953;11: 28–36. doi: 10.1016/0006-3002(53)90005-5 13066452

29. Lohmann W, Fowler CF, Moss AJ Jr., Perkins WH. Some remarks about the effect of glycerol on cells during freezing and thawing: electron-spin resonance investigations concerning this effect. Experientia. 1964;20: 290. doi: 10.1007/bf02151816 4285443

30. Bredderman PJ, Foote RH. Volume of stressed bull spermatozoa and protoplasmic droplets, and the relationship of cell size to motilityd fertility. J Anim Sci. 1969;28: 496–501. doi: 10.2527/jas1969.284496x 5779237

31. Gao DY, Lin S, Watson PF, Critser JK. Fracture phenomena in an isotonic salt solution during freezing and their elimination using glycerol. Cryobiology. 1995;32: 270–284. doi: 10.1006/cryo.1995.1026 7781329

32. Graham EF, Vogt DW, Fisher GR. Effect of method of glycerol addition on the fertility of frozen bovine spermatozoa. J Dairy Sci. 1958;41: 1553–1556.

33. Anzar M, Kroetsch T, Boswall L. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. Anim Reprod Sci. 2011;126: 23–31. doi: 10.1016/j.anireprosci.2011.04.018 21621352

34. Prentice-Biensch JR, Singh J, Mapletoft RJ, Anzar M. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development. Reprod Biol Endocrinol. 2012;10: 73. doi: 10.1186/1477-7827-10-73 22954348

35. Rosenkrans CF Jr., Zeng GQ, GT MC, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biol Reprod. 1993;49: 459–462. doi: 10.1095/biolreprod49.3.459 8399836

36. Nolan JP, Hammerstedt RH. Regulation of membrane stability and the acrosome reaction in mammalian sperm. FASEB J. 1997;11: 670–682. doi: 10.1096/fasebj.11.8.9240968 9240968

37. Sieme H, Oldenhof H, Wolkers WF. Sperm membrane behaviour during cooling and cryopreservation. Reprod Dom Anim. 2015;50 Suppl 3: 20–26.

38. Boughter CT, Monje-Galvan V, Im W, Klauda JB. Influence of cholesterol on phospholipid bilayer structure and dynamics. J Phys Chem B. 2016;120: 11761–11772. doi: 10.1021/acs.jpcb.6b08574 27771953

39. Anzar M, Hassan MM, Graham EF, Deyo RC, Singh G. Efficacy of the Hamilton Thorn Motility Analyzer (HTM-2030) for the evaluation of bovine semen. Theriogenology. 1991;36: 307–317. doi: 10.1016/0093-691x(91)90388-t 16727002

40. Polge C. Fertilizing capacity of bull spermatozoa after freezing at 79o C. Nature. 1952;169: 626–627.

41. Wundrich K, Paasch U, Leicht M, Glander HJ. Activation of caspases in human spermatozoa during cryopreservation—an immunoblot study. Cell and tissue banking. 2006;7: 81–90. doi: 10.1007/s10561-005-0276-7 16732410

Článek vyšel v časopise


2019 Číslo 10