Skip to main content Accessibility help
×
Home

Development and viability of bovine preimplantation embryos after the in vitro infection with bovine herpesvirus-1 (BHV-1): immunocytochemical and ultrastructural studies

  • A.V. Makarevich (a1) (a2), J. Pivko (a1), E. Kubovicova (a1), P. Chrenek (a1), M. Slezaková (a2) and F. Louda (a2)...

Summary

The aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 106.16 TCID50/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR–FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles.

It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.

Copyright

Corresponding author

All correspondence to: A.V. Makarevich, Slovak Agricultural Research Centre, Hlohovska 2, Nitra, Slovak Republic. Tel: +421 37 6546 335. Fax: +421 37 6546 480. e-mail: makarevic@scpv.sk

References

Hide All
Bielanski, A. & Dubuc, C. (1993). In vitro fertilization of bovine oocytes exposed to bovine herpesvirus-1 (BHV-1). Reprod. Domest. Anim. 28, 285–88.
Bielanski, A. & Dubuc, C. (1994). In vitro fertilization and culture of ova from heifers infected with bovine herpesvirus-1 (BHV-1). Theriogenology 41, 1211–17.
Bielanski, A., Lutze-Wallace, C., Sapp, T. & Jordan, L. (1997). The efficacy of trypsin for disinfection of in vitro fertilized bovine embryos exposed to bovine herpesvirus-1. Anim. Reprod. Sci. 47, 18.
Bowen, R.A., Elsden, R.P. & Seidel, Jr, G.E. (1985). Infection of early bovine embryos with bovine herpesvirus-1. Am. J. Vet. Res. 46, 1095–97.
D'Angelo, M., Piatti, R.M., Richtzenhain, L.J., Visintin, J.A. & Tomita, SY. (2002). Evaluation of trypsin treatment on the inactivation of bovine herpesvirus type I (BHV-1) in experimentally infected bovine embryos originated from in vitro fertilization. Theriogenology 57, 569 (abstract).
Edens, M.S.D., Galik, P.K., Riddell, K.P., Givens, M.D., Stringfellow, D.A. & Loskutoff, N.L. (2003). Bovine herpesvirus-1 associated with single, trypsin-treated embryos was not infective for uterine tubal cells. Theriogenology 60, 1495–504.
Gillespie, J.H., Schlafer, D.H., Foote, R.H., Quick, S., Dougherty, E., Schiff, E. & Allen, S. (1990). Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure. Dtsch. Tierarztl. Wochenschr. 97, 6568.
Guerin, B., Le Guienne, B., Allietta, M., Harlay, T. & Thibier, M. (1990). Effects de la contamination par le BHV-1 sur la maturation et fecundation in vitro des ovocytes des bovines. Rec. Med. Vet. 66, 911–17.
Schlafer, D.H., Gillespie, J.H., Foote, R.H., Quick, S., Pennow, N.N., Dougherby, E.P., Schiff, E.I., Allen, S.E., Powers, P.A.Hall, C.E., et al. (1990). Experimental transmission of bovine viral diseases by insemination with contaminated semen or during embryo transfer. Dtsch. Tierarzel. Wochenschr. 97, 6872.
Singh, E.L., Thomas, F.C., Papp-Vid, G., Eaglesome, V.D. & Hare, W.C. (1982). Embryo transfer as a means of controlling the transmission of viral infections. II. The in vitro exposure of preimplantation bovine embryos to infectious bovine rhinotracheitis virus. Theriogenology 18, 133140.
Stringfellow, D. Recommendations for the sanitary handling of in-vivo-derived embryos. (1998). International Embryo Transfer Society (IETS). In: Stringfellow, D.A. & Seidel, S.M., (eds). Manual of the International Embryo Transfer Society: a Procedural Guide and General Information for the Use of Embryo Transfer Technology, Emphasising Sanitary Procedures. 3rd ed. Savoy, IL: International Embryo Society Inc.
Thibier, M. & Nibart, M. (1987). Disease control and embryo imports. Theriogenology 27, 3747.
Vanroose, G., Nauwynck, H., Van Soom, A., Vanopdenbosch, E. & de Kruif, A. (1996). Comparison of the susceptibility of in vitro produced hatched blastocysts for an infection with bovine herpesvirus-1 and bovine herpesvirus-4. In: Proc. 12th Reunion of European Embryo Transfer Association, Abstract 206.
Vanroose, G., Nauwynck, H., Van Soom, A., Vanopdenbosch, E. & de Kruif, A. (1997). Susceptibility of zona-intact and zona-free in in vitro produced bovine embryos at different stages of development to infection with bovine herpesvirus-1. Theriogenology 47, 1389–402.
Vanroose, G., Nauwynck, H., Van Soom, A., Vanopdenbosch, E. & de Kruif, A. (1999). Effect of bovine herpesvirus-1 or bovine viral diarrhoea virus on development of in vitro produced bovine embryos. Mol. Reprod. Dev. 54, 255–63.
Vanroose, G., Nauwynck, H., Van Soom, A., Ysebaert, M.T., Charlier, G., Van Oostreldt, P. et al. (2000). Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biol Reprod. 62, 463–69.
Wright, J. (1998) Photomicrographic illustration of embryo codes. In: Stringfellow, D.A. & Seidel, S.M. (eds) IETS Manual. 3rd ed. Savory, IL: International Embryo Society. Inc, pp. 167–70.

Keywords

Related content

Powered by UNSILO

Development and viability of bovine preimplantation embryos after the in vitro infection with bovine herpesvirus-1 (BHV-1): immunocytochemical and ultrastructural studies

  • A.V. Makarevich (a1) (a2), J. Pivko (a1), E. Kubovicova (a1), P. Chrenek (a1), M. Slezaková (a2) and F. Louda (a2)...

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.