Document Type : Original Article


1 Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Animal Biotechnology, Faculty of Agriculture Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

3 Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

4 Department of Animal Virology, Research and Diagnosis, Razi Vaccine and Serum Research Institute (AREEO), Karaj, Iran

5 Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran


Bovine viral diarrhea virus (BVDV) is an important viral agent causing reproductive failure in cattle. The objectives of the current study were to investigate the interaction between two BVDV biotypes, cytopathic (CP) and Non-cytopathic (NCP) and bovine gametes during in vitro fertilization (IVF) processing, the existence of the virus within embryonic cells and early embryonic development rates. Sperm and ova were exposed separately to CP and NCP BVDV at two concentrations of 104.5 and 105.5 tissue culture infectious dose 50.00% (TCID50) mL-1 prior to IVF, respectively. After five days post-IVF, early embryonic development rates of infected groups were assessed. Several embryos of each group, normal and degenerated, were selected for a viral assay using reverse transcription polymerase chain reaction technique. The result showed that the early embryonic development rates were decreased in treatment groups. The rates in the CP groups were lower than the NCP groups. In the CP groups, the proportions were, respectively, 10.00, 6.00 and 11.00, and 6.00% in the infected sperm and oocyte groups (104.5 and 105.5 TCID50 mL-1) that were higher than 50.00% in the control group. In NCP groups, the rates were, respectively, 25.00, 18.00 and 24.00, and 21.00% in the infected groups compared to 48.00% in the control group. In the CP groups, no BVDV was detected in normal embryos, whereas, all degenerated embryos were completely virus-positive. In the NCP groups, the virus was detected in both normal and degenerated embryos. In conclusion, this study supported detrimental impacts of CP and NCP BVDV on early embryonic development and the role of sperm and the zona pellucida layer as carriers of the virus. 


  1. Hopper RM, King EH. Evaluation of breeding soundness: Basic examination of the semen.In: Hopper RM (Ed). Bovine reproduction. New Jersey, USA: John Wiley & Sons, Inc. 2014; 68-78.
  2. Yavru S, Kale M, Gulay M, et al. Effects of bovine viral diarrhoea virus on the fertility of cows. Acta Vet Hung 2013; 61(2): 281-289.
  3. Van Soom A, Wrathall AE, Herrler A, et al. Is the zona pellucida an efficient barrier to viral infection? Reprod Fertil Dev 2010; 22(1): 21-31.
  4. Garoussi MT, Mehrzad J. Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle. Theriogenology 2011; 75(6): 1067-1075.
  5. Gard JA, Givens MD, Stringfellow DA. Bovine viral diarrhea virus (BVDV): epidemiologic concerns relative to semen and embryos. Theriogenology 2007; 68(3): 434-442.
  6. Gard JA. Research challenges involving embryo patho-gen interactions. Anim Reprod 2015; 12(3): 444-449.
  7. Vanroose G, Nauwynck H, Van Soom A, et al. Replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona-free and zona-intact in vitro-produced bovine embryos and the effect on embryo quality. Biol Reprod 1998; 58(3): 857-866.
  8. Thibier M, Wagner HG. World statistics for artificial insemination in cattle. Livest Prod Sci 2002; 74(2): 203-212.
  9. Stroud B. IETS 2011 statistics and data retrieval committee report. The year 2010 worldwide statistics of embryo transfer in domestic farm animals. Available at: Accessed 02 Feb, 2023.
  10. Lindberg A, Ortman K, Alenius S. Seroconversion to bovine viral diarrhea virus (BVDV) in dairy heifers after embryo transfer. In proceedings: 14th International congress on animal reproduction. Stockholm, Sweden 2000; 250.
  11. Nettleton PF, Gunn G. BVD virus genotype 2 in British cattle. Vet Rec 2002; 151(20): 616.
  12. Vilcek S, Herring AJ, Herring JA, et al. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 1994; 136(3-4): 309-323.
  13. Deregt D, Smithson S, Kozub GC. A short incubation serum neutralization test for bovine viral diarrhea virus. Can J Vet Res 1992; 56(2): 161-164.
  14. Fay PC, Cook CG, Wijesiriwardana N, et al. Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus. J Virol Methods 2020; 285: 113943. doi: 10.1016/j.jviromet.2020.113943.
  15. Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938; 27(3): 493-497.
  16. Al-Essawe E, Wallgren M, Wulf M, et al. Addition of seminal plasma reduces binding of stallion spermatozoa to bovine oocytes. In proceedings: 33rd meeting of the association of embryo transfer in Europe. Bath, UK 2017; 910.
  17. Garoussi MT. The effects of cytopathic and noncytopathic bovine viral diarrhoea virus with sperm cells on in vitro fertilization of bovine oocytes. Vet Res Commun 2007; 31(3): 365-370.
  18. Ponsart C, Pozzi N. Sanitary requirements for bovine gametes and embryos in international trade. Anim Reprod 2013; 10(3): 283-296.
  19. Madison V, Avery B, Greve T. Selection of immature bovine oocytes for developmental potential in vitro. Anim Reprod Sci 1992; 27(1): 1-11.
  20. Magdanz V, Boryshpolets S, Ridzewski C, et al. The motility-based swim-up technique separates bull sperm based on differences in metabolic rates and tail length. PLoS One 2019; 14(10): e0223576. doi:10.1371/journal.pone.0223576.
  21. Wei Y, Idrees M, Sidrat T, et al. BOEC–Exo addition promotes in vitro maturation of bovine oocyte and enhances the developmental competence of early embryos. Animals (Basel) 2022; 12(4): 424. doi: 10.3390/ani12040424.
  22. Saini S, Ansari S, Sharma V, et al. Folate receptor-1 is vital for developmental competence of goat embryos. Reprod Domest Anim 2022; 57(5): 541-549.
  23. Afshar A, Dulac GC, Dubuc C, et al. Comparative evaluation of the fluorescent antibody test and microtiter immunoperoxidase assay for detection of bovine viral diarrhea virus from bull semen. Can J Vet Res 1991; 55(1): 91-93.
  24. Okazaki K, Honda E, Kono Y. Heparin-binding domain of bovid herpesvirus 1 glycoprotein gIII. Arch Virol 1994; 134(3-4): 413-419.
  25. Zanrosso D. The hidden risk: transmission of pathogens micro by mean of embryo transfer in cattle [Italian]. Bachelor Thesis. University of Parma, Parma, Italy: 2019.
  26. Maertens A, Bourlet T, Plotton N, et al. Validation of safety procedures for the cryopreservation of semen contaminated with hepatitis C virus in assisted reproductive technology. Hum Reprod 2004; 19(7): 1554-1557.
  27. Morfeld KA, White B, Mills G, et al. 186 A novel method for eliminating porcine reproductive and respiratory syndrome virus from boar semen and its effects on embryo development. Reprod Fertil Dev 2004; 17: 243-243.
  28. Boccaccio A, Frassanito MC, Lamberti L, et al. Nanoscale characterization of the biomechanical hardening of bovine zona pellucida. J R Soc Interface 2012; 9(76): 2871-2882.
  29. Bielanski A, Dubuc C. In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus. Anim Reprod Sci 1995; 38(3): 215-221.
  30. Blondin P. Health and safety of IVF embryos: challenges for the international ET industry. Anim Reprod 2014; 11(3): 270-277.
  31. Queiroz-Castro VLD, da Costa EP, Alves SVP, et al. Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection. PLoS One 2019; 14: e0218963. doi: 10.1371/journal. pone.0218963.
  32. Wrathall AE, Simmons HA, Van Soom A. Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen. Theriogenology 2006; 65(2): 247-274.
  33. Brodersen BW. Bovine viral diarrhea virus infections: manifestations of infection and recent advances in understanding pathogenesis and control. Vet Pathol 2014; 51(2): 453-464.
  34. Ambrose RK, Gravel JL, Commins MA, et al. In vivo characterisation of five strains of bovine viral diarrhoea virus 1 (Subgenotype 1c). Pathogens 2018; 7(1): 12. doi: 10.3390/pathogens7010012.
  35. Suzuki H, Yang X, Foote RH. Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro. Mol Reprod Dev 1994; 38(4): 421-430.
  36. Vanroose G, Nauwynck H, Van Soom A, et al. Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biol Reprod 2000; 62(2): 463-469.
  37. Stringfellow DA, Givens MD. Infectious agents in bovine embryo production: hazards and solutions. Theriogenology 2000; 53(1): 85-94.