Supplementation of biotin to sperm preparation medium increases the motility and longevity in cryopreserved human spermatozoa

  • Guruprasad Kalthur
  • Sujith R Salian
  • Farid Keyvanifard
  • Shyam Sreedharan
  • Jeena S Thomas
  • Pratap Kumar
  • Satish K Adiga
Gamete Biology



To study the effect of supplementing biotin to sperm preparation medium on the motility of frozen-thawed spermatozoa.


Semen samples of men attending the University infertility clinic (n = 105) were cryopreserved using glycerol-egg yolk-citrate buffered cryoprotective medium in liquid nitrogen. After a period of two weeks, the semen samples were thawed and the motile spermatozoa were extracted by swim-up technique using Earle’s balanced salt solution (EBSS) medium supplemented with either biotin (10 nM) or pentoxifylline (1 mM). The post-wash motility was observed up to 4 h after incubation.


Both biotin and pentoxifylline supplementation resulted in significant increase in total motility (p < 0.05), progressive motility (p < 0.001) and rapid progressive motility (p < 0.05 v/s biotin and p < 0.01 v/s pentoxifylline) compared to the control at 1 h post-incubation period. Significantly higher percentage of total (p < 0.01, p < 0.05 in biotin and pentoxifylline respectively), progressive (p < 0.001) and rapid progressive motilities (p < 0.01) were observed in these two groups even at 2 h compared to the control. In the control group at 4 h after incubation, ~11% decline in total motility and ~8% decline in progressive motility was observed. However, in both biotin and pentoxifylline group the motility was significantly higher than control (p < 0.001). No significant difference in the motility was observed between biotin and pentoxifylline groups at any of the time intervals studied.


Biotin can enhance the sperm motility and prolong the survival of frozen-thawed semen samples which may have potential benefit in assisted reproductive technology field.


Biotin pentoxifylline spermatozoa cryopreservation post-thaw motility 



Authors kindly acknowledge the technical support provided by Mrs. Jalyalaxmi Pai, Mrs. Kirthi Patil, and Ms. Anita Poojary. Authors thank Dr. Prashanth Naik and Mr. Naveenchandra Kumar, Mangalore University for editing the language of the manuscript.


  1. 1.
    Stanic P, Sonicki Z, Suchanek E. Effect of pentoxifylline on motility and membrane integrity of human cryopreserved spermatozoa. Int J Androl. 2002;25:186–90.PubMedCrossRefGoogle Scholar
  2. 2.
    Tournaye H, Van der Linden M, Van den Abbeel E, Devroey P, Van Steirteghem A. Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos. Hum Reprod. 1993;8:1475–80.PubMedGoogle Scholar
  3. 3.
    Tournaye H, Van der Linden M, Van den Abbeel E, Devroey P, Van Steirteghem A. Mouse in vitro fertilization using sperm treated with pentoxifylline and 2-deoxyadenosine. Fertil Steril. 1994;62:644–7.PubMedGoogle Scholar
  4. 4.
    Scott L, Smith S. Human sperm motility enhancing agents have detrimental effect on mouse oocytes and embryos. Fertil Steril. 1995;63:166–75.PubMedGoogle Scholar
  5. 5.
    Mock DM. Marginal biotin deficiency is common in normal human pregnancy and is highly teratogenic in mice. J Nutr. 2009;139:154–7.PubMedGoogle Scholar
  6. 6.
    Watanabe T. Dietary biotin deficiency affects reproductive function and prenatal development in hamsters. J Nutr. 1993;123:2101–8.PubMedGoogle Scholar
  7. 7.
    World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge: Cambridge University Press; 1999.Google Scholar
  8. 8.
    Kovacic B, Vlaisavljevic V, Reljic M. Clinical use of pentoxifylline for activation of immotile testicular sperm before ICSI in patients with non-obstructive azoospermia. J Androl. 2006;27:45–52.PubMedCrossRefGoogle Scholar
  9. 9.
    Kalthur G, Adiga SK, Upadhya D, Rao S, Kumar P. Effect of cryopreservation on sperm DNA integrity in patients with teratospermia. Fertil Steril. 2008;89:1723–7.PubMedCrossRefGoogle Scholar
  10. 10.
    Mahadevan M, Baker G. Assessment and preparation of semen for in vitro fertilization. In: Wood C, Trounson A, editors. Clinical in vitro fertilization. Berlin: Springer; 83. p. 97–1984.Google Scholar
  11. 11.
    Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. The effect of cryoprotectants on motility. Fertil Steril. 1988;50:314–20.PubMedGoogle Scholar
  12. 12.
    Kalthur G, Raj S, Thiyagarajan A, Kumar S, Kumar P, Adiga SK. Vitamin E supplementation in semen cryopreservation medium improves the motility and protects sperm from freeze-thaw-induced DNA damage. Fertil Steril. 2011;95:1149–51.PubMedCrossRefGoogle Scholar
  13. 13.
    Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril. 1990;53:715–22.PubMedGoogle Scholar
  14. 14.
    Yovich JL. Pentoxifylline: actions and applications in assisted reproduction. Hum Reprod. 1993;8:1786–91.PubMedGoogle Scholar
  15. 15.
    Yunes R, Fernadez P, Doncel GF, Acosta AA. Cyclin nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa. Biocell. 2005;29:287–93.PubMedGoogle Scholar
  16. 16.
    Gil MA, Hernandez M, Roca J, et al. Pentoxifylline added to freezing or post-thaw extenders does not improve the survival or in vitro fertilizing ability of boar spermatozoa. Reproduction. 2010;139:557–64.PubMedCrossRefGoogle Scholar
  17. 17.
    Centola GM, Cartie RJ, Cox C. Differential responses of human sperm to varying concentrations of pentoxifylline with demonstration of toxicity. J Androl. 1995;16:136–42.PubMedGoogle Scholar
  18. 18.
    Tash JS, Means AR. Cyclic adenosine 3′,5′ monophosphate, calcium and protein phosphorylation in flagellar motility. Biol Reprod. 1983;28:75–104.PubMedCrossRefGoogle Scholar
  19. 19.
    Karl PI, Fisher SE. Biotin transport in microvillous membrane vesicles, cultured trophoblasts and the isolated perfused cotyledon of the human placenta. Am J Physiol. 1992;262:C302–8.PubMedGoogle Scholar
  20. 20.
    Schenker S, Hu Z, Johnson RF, Yang Y, Frosto T, Elliott BD, Henderson GI, Mock DM. Human placental biotin transport: normal characteristics and effect of ethanol. Alcohol Clin Exp Res. 1993;17:566–75.PubMedCrossRefGoogle Scholar
  21. 21.
    Feldman G, Wolf B. Deficient acetyl CoA carboxylase activity in multiple carboxylase deficiency. Clin Chim Acta. 1981;111:147–51.PubMedCrossRefGoogle Scholar
  22. 22.
    Packman S, Caswell N, Gonzales-Rios M, Kadlecek T, Cann H, Rassin D, McKay C. Acetyl CoA carboxylase in cultured fibroblasts: differential biotin dependence in the two types of biotin-responsive multiple carboxylase deficiency. Am J Hum Genet. 1984;36:80–92.PubMedGoogle Scholar
  23. 23.
    Kuroishi T, Rios-Avila L, Pestinger V, Wijeratne SS, Zempleni J. Biotinylation is a natural, albeit rare, modification of human histones. Mol Genet Metab. 2011;104:537–45.PubMedCrossRefGoogle Scholar
  24. 24.
    Atamna H, Newberry J, Erlitzki R, Schultz CS, Ames BN. Biotin deficiency inhibits heme synthesis and impairs mitochondria in human lung fibroblasts. J Nutr. 2007;137:25–30.PubMedGoogle Scholar
  25. 25.
    Zempleni J, Teixeira DC, Kuroishi T, Cordonier EL, Baier S. Biotin requirements for DNA damage prevention. Mutat Res 2011;doi: mrfmmm2011.08.001/mrfmmm 2011.08.001.
  26. 26.
    Zempleni J, Mock DM. Marginal biotin deficiency is teratogenic. Proc Soc Exp Biol Med. 2000;223:14–21.PubMedCrossRefGoogle Scholar
  27. 27.
    Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992;327:1832–5.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Guruprasad Kalthur
    • 1
    • 2
  • Sujith R Salian
    • 1
  • Farid Keyvanifard
    • 1
  • Shyam Sreedharan
    • 1
  • Jeena S Thomas
    • 1
  • Pratap Kumar
    • 1
  • Satish K Adiga
    • 1
  1. 1.Clinical Embryology, Division of Reproductive Medicine, Kasturba Medical CollegeManipal UniversityManipalIndia
  2. 2.Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical CollegeManipal UniversityManipalIndia

Personalised recommendations