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In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus
ANIMAL REPRODUCTION SCIENCE E LS EV I ER
Animal Reproduction Science 38 ( 1995 ) 215-221
In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus A. Bielanski*, C. D u b u c Agriculture Canada, Animal Diseases Research Institute, Nepean, P.O. Box 11300, Ont., Canada K2H 8P9
Accepted 14 September 1994
Abstract Bovine viral diarrhea virus infection was induced in 16 heifers by inoculation of a noncytopathic strain of bovine viral diarrhea virus (BVDV). Six BVDV-free heifers served as controls. On Day 8 after inoculation, cumulus-oocyte complexes were collected from ovaries of animals at the second peak of fever preceded by leukopenia. The oocytes were then matured and fertilized in vitro. There was no significant difference (48% vs. 54% P> 0.05 ) in the percentage of cleaved oocytes between infected and non-infected animals. However, the proportion of embryos that developed to the blastocyst stage was significantly higher for the control group than for BVDV group (29% vs. 14%) (P< 0.01 ). All follicular fluids and cumulus-oocyte complexes collected from infected animals tested positive for the presence of the virus, but embryos produced by in vitro fertilization 7 days after in vitro co-culture tested negative. Keywords: Bovine viral diarrhea virus; Embryo; Fertilization; Ova
I. Introduction Bovine viral diarrhea virus ( B V D V ) is a pathogen o f cattle causing significant worldwide economic losses for the cattle industry (Radostis and Littlejohns, 1988 ). Infection with B V D V coinciding with the time o f insemination is associated with decreased fertilization and conception rates ( G r a h n et al., 1984; Virakul et al., 1988; M c G o w a n et al., 1993). Recently, B V D V has been found in * Corresponding author. 0378-4320/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10378-4320 (94) 1358-6
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A. Bielanski, C. Dubuc /Animal Reproduction Science 38 (1995)215-221
the follicular fluids and oviductal cells of commercially slaughtered cows used for the in vitro production of embryos (Bielanski et al., 1993 ). The objective of this paper was to study the consequences of experimental BVDV infection on embryos fertilized in vitro.
2. Materials and methods
Sixteen BVDV seronegative and virus-free (from NADL, Singer, and New York strains) heifers were injected intravenously with 5 ml of BVDV (107 TCIDso ml-1, non-cytopathic New York 1 strain, National Veterinary Service Laboratory, Ames, IA, USA). Six BVDV-free heifers injected with the physiological saline served as controls. All the heifers were bled before injection and then every second day until day of slaughter, and blood cell counts were determined. Oocytes were obtained from ovaries of the animals at slaughter on Day 8 after injection, which coincided with a second peak of fever preceded by leukopenia. This approach was to assure exposure of ova to high concentrations of the ¥irus in follicular fluid before the possibility of subsequent antibody formation and virus neutralization. 2.1. Collection and maturation of oocytes
Ovaries were collected from cattle within minutes after slaughter, washed twice in Dulbecco's phosphate-buffered saline (D-PBS) and then processed immediately in the laboratory. Follicular fluids were aspirated from antral follicles (2-7 mm in diameter, regardless of the stage follicular atresia) using an 18 gauge needle attached to a syringe and released into a centrifuge tube. After 10-15 min, the supernatant fluids were removed and cumulus-oocyte complexes were collected from the sediment, washed twice in D-PBS and once in maturation medium. All oocytes, except those with only a few attached cumulus cells and those with cumulus cells scattered in dark clumps in a jelly matrix, were cultured. The maturation medium was TCM-199 with Earle's salts (Gibco Laboratories, Grand Island, NY) supplemented with 20% estrus cow serum (free of antibodies to BVDV and free of BVDV), 0.4 mM L-glutamine (Sigma Chemical Co., St. Louis, MO), 0.2 mM Na-pyruvate (Sigma), and 50/tg ml-1 gentamicin (Sigma). Oocytes from each cow in groups of 5-10 were matured for 24 h in 50/tl drops of maturation medium overlaid with silicone oil (Dow Coming, Midland, MI) in culture dishes (Nunc, InterMed, Kamstrup, Denmark) at 38.5 °C and 5% CO2 in air with 100% humidity for 24 h. 2.2. Preparation of oviductal cells
Bovine oviducts from the six control heifers were collected and used for in vitro fertilization (IVF) and co-culture. Oviductal cell suspension cultures were prepared and used as described previously (Bielanski and Loewen, 1994).
A. Bielanski, C. Dubuc / Animal Reproduction Science 38 (1995) 215-221
217
2.3. In vitro fertilization of oocytes Semen pooled from five bulls free of BVDV was used. Spermatozoa for IVF were prepared by a swim-up method described by Parrish et al. ( 1986 ). Briefly, thawed semen (1 ml) was pipetted under 1.5 ml of modified Tyrode's solution containing 25 mM of hepes buffer (TALP-HEPES) in 5 ml tubes and held for 75 min at 38.5 °C. After incubation, 0.8 ml of the supernatant was aspirated from each tube and pooled. The sample was centrifuged at 200g for 10 rain and the supernatant was removed. The pellet was then washed once more by centrifugation with 10 ml of fresh TALP-HEPES medium prior to being reconstituted and used for insemination. After maturation, oocytes were washed three times in TALP-HEPES and once in fertilization medium (IVF-TALP). The oocytes were inseminated in IVF drops (50 #1) with approximately 50 × 105 of motile spermatozoa (pool of five BVDVnegative bulls) and then incubated at 38.5 °C in 5% CO2 in air for 18 h.
2.4. Embryo culture After IVF, the oocytes were freed from cumulus cells by repeated passage through a small-bore pipet and washed twice in D-PBS medium and once in coculture medium. The co-culture medium was INRA Menezo-B2 (Bio Merieux, Paris) supplemented with 1/A BOEC, 10% estrus cow serum (free of antibodies to BVDV and free of BVDV) and 50 #g ml- ~ gentamicin. After washing, 5-10 zygotes were placed in each droplet of co-culture medium and incubated under silicone oil (360 Medical Fluid, Dow Corning, Midland, MI) at 38.5°C in 5% CO2 in air for 8 days. The cleavage rate of oocytes was determined 72 h after insemination, and was calculated as the proportion of eggs which developed to at least the two-cell stage. At the end of incubation, the numbers of embryos that developed to morula and blastocyst stages were determined by direct microscopic examination. The washing procedure used in this study refers to the transfer of oocytes/embryos by a capillary glass pipet in approximately 3/zl medium to a clean dish containing 2 ml of fresh medium, and then gently swirling the dish and embryos for a few seconds. For each transfer a new pipet was used.
2.5. Virus assay The following samples were collected for viral assay: blood serum, follicular fluid, oviductal cells, uterine wash-fluid, granulosa cells, cumulus-oocyte complexes and embryos. The embryos (in groups of 2-5) and oviductal cells were disrupted by sonication (Ultrasonic Processor, Model GE-600, Manostat, New York, NY, USA) for 40 s at 4 °C prior to being assayed. The uterine wash fluid was obtained by washing uterine horn with 20 ml of D-PBS. All samples were frozen in cryovials and stored at - 70 ° C until assayed.
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2.6. Viral detection and titration The immunoperoxidase staining technique (Afshar et al., 1991 ) was used for BVDV detection and titration of samples. Titration was done by making ten-fold dilutions (undiluted to 10-5) in Earlie's minimum essential medium (MEM (E) ) in quadruplicate. Ten microliters of each dilution was used with a suspension of bovine turbinate (BT) cells (3 × 105 m l - ) in a tissue culture microtiter plate. After incubation for 24 h, monolayer cell cultures were fixed with 20% acetone and stained by the addition of bovine anti-BVD serum followed by a horseradish peroxidase labeled rabbit antibovine IgG conjugate. Excess conjugate was then removed by washing, following which the enzyme substrate was added and permitted to incubate until the desired staining intensity was reached. Positive cells exhibit reddish brown cytoplasmic staining in response to the precipitating substrate, whereas non-reacting cells remain clear and colorless when viewed by light microscope. Proper controls were included in each test as follows: the BT were tested with bovine anti-BVD serum, the BVDV-infected BT cells were tested with both a negative serum (BVD-antibodies free) and the bovine anti-BVD serum, and the conjugate was reacted with the BVDV-infected BT cells. Titrations were calculated by the Reed and Munch method (Davis et al., 1990). When samples were found negative with the microtiter plate immunoperoxidase test a passage was made in a flask. Briefly, bovine kidney cells, second to fifth passage, were grown in a 25 ml Falcon flask (Becton Dickinson, Lincoln Park, NJ ) in MEM (E), supplemented with 10% FCS and antibiotics. The cells were monitored routinely for BVDV; the FCS was pretested for BVDV and anti-BVDV antibody and treated with 1 Mrad of gamma irradiation at 0-4 ° C. The monolayers were washed with MEM(E), and the flask was inoculated with the sample (500/tl) to be tested. After an absorption period of 1 h at 37°C on a rocker, the cultures were maintained in MEM(E) containing 2% FCS for 5 days at 37°C. Cell cultures inoculated with BVDV samples were tested for virus antigens on Day 4 after inoculation using the immunoperoxidase test as described above.
2. 7. Statistical method The proportion of fertilized ova that cleaved, and the proportion of fertilized ova that developed to the blastocyst stage were evaluated by the Z2 test. Differences at a probability of less than 0.05 were considered significant.
3. Results
All BVDV-injected animals had biphasic fever (40-41.5 ° C ) on Days 3-4 and again Day 8 post inoculation. In addition, leukopenia accompanied fever on Days 3-4. BVDV was isolated from the sera of 15/16 and 4/16 animals on Day 4 and Day 8 post-injection, respectively. In total, 71 cumulus-oocyte complexes, 130 one-cell ova and 120 embryos obtained from infected heifers were tested for
A. BielanskL C. Dubuc / Animal Reproduction Science 38 (1995) 215-221
219
Table 1 BVD isolation from the samples of BDV-injected animals Samples
No. of positive animals
Viral trier (rage) (TCIDso ml- ~)
Follicular fluid Granulosa cells Oviductal cells Uterine wash-fluid Cumulus-oocyte complexes IVF embryos
16/ 16 16/ 16 0/16 4/16 16/16 0/16
103-107 102-106 Negative Positive- l 03 103-105 Negative
TCIDso - Medium tissue culture infectious dose.
BVDV. Results of virus isolation from BVDV-injected heifers are presented in Table 1. None of the non-injected animals showed any significant changes in rectal temperature (37.5-38.5 °C) or leukopenia in the blood. Also, all samples from control animals tested negative on virus isolation. From a total of 329 oocytes collected from infected animals, 258 were inseminated and cultured. A mean cleavage percentage of 47.6% (123/258) was obtained vs. 53.9% (62/115 ) for control non-infected oocytes (P> 0.05 ). The proportion of embryos that developed to blastocyst stages was significantly higher (P< 0.01 ) for the control group (29%, 18/62)than for the BVDV group (13.8%, 17/123).
4. Discussion
Inoculation of animals with BVDV induced an acute stage of disease similar to that described by Radostis and Littlejohns (1988), Corapi et al. (1989) and Brownlie (1990). It was clearly demonstrated in this experiment that the oocytes were exposed to a high titer of virus by contact with contaminated follicular fluid and the granulosa cells surrounding them in the ovaries. However, embryos produced by IVF from such oocytes were not associated with infectious virus. A number of manipulations (removing cumulus cells and washing) as well as prolonged in vitro culturing of embryos at 39 °C could account for virus inactivation or its removal. It was established previously that a simple washing procedure is sufficient to remove the virus if present at low titre ( 104-105 TCIDso m l - 1) from the intact zona pellucida of uterine stage embryos (Singh et al., 1982). Oviductal cell are in common use for co-culture of IVF-produced embryos, and may be a source of viral infection (Bielanski et al., 1993). It is surprising and difficult to explain why in the present experiment all the infected animals yielded negative oviductal cells but some had infected uterine flushes. Recently, it has been demonstrated that cows persistently infected with BVDV are poor embryo producers, but can yield uterine stage embryos which develop into non-infected offspring using embryo transfer (Wentink et al., 1991; Bak et al., 1992 ). Also, embryos produced by IVF with BVDV-infected semen from per-
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A. Bielanski, C. Dubuc / Animal Reproduction Science 38 (I 995) 215-221
sistently infected bulls were not associated with the virus (Bielanski and Loewen, 1994). Collectively, the results as presented in this paper and reported by others indicate that the risk of transmission of BVDV through embryos fertilized in vitro is minimal provided the embryos are washed according to the procedures recommended by the International Embryo Transfer Society (Anonymous, 1990). The proportion of oocytes that cleaved from infected animals was only slightly lower than those obtained from noninfected animals. This proportion was, however, comparable with the proportion reported when semen from persistently infected bulls was used for IVF of 'clean' oocytes (Bielanski and Loewen, 1994). In contrast, a significant proportion of embryos obtained from infected animals failed to develop to the morula or blastocyst stage. The reason for this is not clearly understood because the virus is not cytopathic for early embryos in vitro (Bielanski and Hare, 1988 ). It is possible that the fever associated with the viremic stage in heifers affected the developmental competence of oocytes, as was suggested previously when oocytes from BHV-1 infected animals were fertilized in vitro (Bielanski and Dubuc, 1993). This theory might also be supported by the reported decresed fertilization and conception rates after experimental infection of heifers with BVDV around the time of insemination (McGowan et al., 1993). Overall, the results of this study indicate that low numbers of viable and transferable-stage embryos may be produced in vitro from BVDV-infected heifers and that such embryos are not associated with the virus. However, the developmental capacity of the embryos in vivo, after transfer to recipients, is still to be determined.
Acknowledgments The technical assistance of G. Raby and P. ChatiUon is gratefully acknowledged.
References Afshar, A., Dulac, G.C., Dubuc, C. and Howard,T.H., 1991. Comparativeevaluationof the fluorescent antibodytest and microtiter immunoperoxidaseassayfor detectionof bovine viral diarrhea virus from bull semen. Can. J. Vet. Res., 55: 91-93. Anonymous, 1990. Reeomendationfor the sanitaryhandlingof embryos. In: D.A. Strigfellowand S.M. Seidel (Editors), Manualof the InternationalEmbryoTransfer Society.IETS, Champaign, IL, pp. 41-45. Bak,A., Callesen,H., Meyling,T. and Greve,T., 1992. Calvesborn afterembryotransferfromdonors persistentlyinfectedwith BVD virus. Vet. Rec., 131: 37. Bielanski,A. and Dubuc, C., 1993. In vitro fertilizationof bovine oocytesexposedto bovine herpesvirus-1 (BHV-1). Reprod. Domest.Anim.,28: 285-288. Bielanski,A. and Hare,W.C.D., 1988.Effectin vitro ofbovineviraldiarrheavirus on bovineembryos with the zona pellucidaintact, damagedand removed.Vet. Res. Commun., 12:19-24. Bielanski, A. and Loewen,K.S., 1994. In vitro fertilizationof bovine oocyteswith semenfrom bulls persistentlyinfectedwithbovine viral diarrheavirus. Anim.Reprod. Sci., 35:183-189. Bielanski, A., Loewen,K.S., Del Campo, R.M., Sirard, M.A. and Willadsen, S., 1993. Isolationof
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bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV) in association with the in vitro production of bovine embryos. Theriogenology, 40:531-538. Brownlie, J., 1990. The pathogenesis of bovine virus diarrhoea virus infections. Rev. Sci. Tech. Off. Int. Epiz., 9; 43-59. Corapi, W.V., French, T.W. and Dubovi, E.J., 1989. Severe trombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus. J. Virol., 63: 3934-3943. Davis, B.D., Dulbecco, R., Eisen, H.N. and Ginsberg, H.S., 1990. Microbiology, 4th edn. Lippincott, Philadelphia, PA, pp. 769-794. Grahn, T.C., Fahning, M.L. and Zemjanis, R., 1984. Nature of early reproductive failure by bovine viral diarrhea virus. J. Am. Vet. Med. Assoc., 185: 429-432. McGowan, M.R., Kirkland, P.D., Richards, S.G. and Littlejohns, I.R., 1993. Increased reproductive losses in cattle infected with bovine pestivirus around the time of insemination. Vet. Rec., 133: 39-43. Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Critzer, E.S., Eyestone, W.H. and First, N.L., 1986. Bovine in vitro fertilization with frozen-thawed sperm. Theriogenology, 25:591-600. Radostis, O.M. and Littlejohns, R. 1988. New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus. Can. Vet. J., 29:513-528. Singh, E.L., Eaglesome, M.D., Thomas, F.C., Papp-Vid, G. and Hare, W.C.D., 1982. Embryo transfer as a means of controlling the transmission of viral infections. I. The in vitro exposure of preimplantation embryos to akabane, blue-tongue and bovine viral diarrhea viruses. Theriogenotogy, 17: 437-444. Virakul, P., Fahning, M.L., Joo, H.S. and Zemjanis, R., 1988. Fertility of cows challenged with a cytopathic strain of bovine viral diarrhea virus during an outbreak of spontaneous infection with a noncytopathic strain. Theriogenology, 29:441-449. Wentink, G.H., Aarts, T., Mirck, M.H. and van Exsel, A.C.A., 1991. Calf from a persistently infected heifer born after embryo transfer with normal immunity to BVDV. Vet. Rec., 129: 449-450.
Animal Reproduction Science 38 ( 1995 ) 215-221
In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus A. Bielanski*, C. D u b u c Agriculture Canada, Animal Diseases Research Institute, Nepean, P.O. Box 11300, Ont., Canada K2H 8P9
Accepted 14 September 1994
Abstract Bovine viral diarrhea virus infection was induced in 16 heifers by inoculation of a noncytopathic strain of bovine viral diarrhea virus (BVDV). Six BVDV-free heifers served as controls. On Day 8 after inoculation, cumulus-oocyte complexes were collected from ovaries of animals at the second peak of fever preceded by leukopenia. The oocytes were then matured and fertilized in vitro. There was no significant difference (48% vs. 54% P> 0.05 ) in the percentage of cleaved oocytes between infected and non-infected animals. However, the proportion of embryos that developed to the blastocyst stage was significantly higher for the control group than for BVDV group (29% vs. 14%) (P< 0.01 ). All follicular fluids and cumulus-oocyte complexes collected from infected animals tested positive for the presence of the virus, but embryos produced by in vitro fertilization 7 days after in vitro co-culture tested negative. Keywords: Bovine viral diarrhea virus; Embryo; Fertilization; Ova
I. Introduction Bovine viral diarrhea virus ( B V D V ) is a pathogen o f cattle causing significant worldwide economic losses for the cattle industry (Radostis and Littlejohns, 1988 ). Infection with B V D V coinciding with the time o f insemination is associated with decreased fertilization and conception rates ( G r a h n et al., 1984; Virakul et al., 1988; M c G o w a n et al., 1993). Recently, B V D V has been found in * Corresponding author. 0378-4320/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10378-4320 (94) 1358-6
216
A. Bielanski, C. Dubuc /Animal Reproduction Science 38 (1995)215-221
the follicular fluids and oviductal cells of commercially slaughtered cows used for the in vitro production of embryos (Bielanski et al., 1993 ). The objective of this paper was to study the consequences of experimental BVDV infection on embryos fertilized in vitro.
2. Materials and methods
Sixteen BVDV seronegative and virus-free (from NADL, Singer, and New York strains) heifers were injected intravenously with 5 ml of BVDV (107 TCIDso ml-1, non-cytopathic New York 1 strain, National Veterinary Service Laboratory, Ames, IA, USA). Six BVDV-free heifers injected with the physiological saline served as controls. All the heifers were bled before injection and then every second day until day of slaughter, and blood cell counts were determined. Oocytes were obtained from ovaries of the animals at slaughter on Day 8 after injection, which coincided with a second peak of fever preceded by leukopenia. This approach was to assure exposure of ova to high concentrations of the ¥irus in follicular fluid before the possibility of subsequent antibody formation and virus neutralization. 2.1. Collection and maturation of oocytes
Ovaries were collected from cattle within minutes after slaughter, washed twice in Dulbecco's phosphate-buffered saline (D-PBS) and then processed immediately in the laboratory. Follicular fluids were aspirated from antral follicles (2-7 mm in diameter, regardless of the stage follicular atresia) using an 18 gauge needle attached to a syringe and released into a centrifuge tube. After 10-15 min, the supernatant fluids were removed and cumulus-oocyte complexes were collected from the sediment, washed twice in D-PBS and once in maturation medium. All oocytes, except those with only a few attached cumulus cells and those with cumulus cells scattered in dark clumps in a jelly matrix, were cultured. The maturation medium was TCM-199 with Earle's salts (Gibco Laboratories, Grand Island, NY) supplemented with 20% estrus cow serum (free of antibodies to BVDV and free of BVDV), 0.4 mM L-glutamine (Sigma Chemical Co., St. Louis, MO), 0.2 mM Na-pyruvate (Sigma), and 50/tg ml-1 gentamicin (Sigma). Oocytes from each cow in groups of 5-10 were matured for 24 h in 50/tl drops of maturation medium overlaid with silicone oil (Dow Coming, Midland, MI) in culture dishes (Nunc, InterMed, Kamstrup, Denmark) at 38.5 °C and 5% CO2 in air with 100% humidity for 24 h. 2.2. Preparation of oviductal cells
Bovine oviducts from the six control heifers were collected and used for in vitro fertilization (IVF) and co-culture. Oviductal cell suspension cultures were prepared and used as described previously (Bielanski and Loewen, 1994).
A. Bielanski, C. Dubuc / Animal Reproduction Science 38 (1995) 215-221
217
2.3. In vitro fertilization of oocytes Semen pooled from five bulls free of BVDV was used. Spermatozoa for IVF were prepared by a swim-up method described by Parrish et al. ( 1986 ). Briefly, thawed semen (1 ml) was pipetted under 1.5 ml of modified Tyrode's solution containing 25 mM of hepes buffer (TALP-HEPES) in 5 ml tubes and held for 75 min at 38.5 °C. After incubation, 0.8 ml of the supernatant was aspirated from each tube and pooled. The sample was centrifuged at 200g for 10 rain and the supernatant was removed. The pellet was then washed once more by centrifugation with 10 ml of fresh TALP-HEPES medium prior to being reconstituted and used for insemination. After maturation, oocytes were washed three times in TALP-HEPES and once in fertilization medium (IVF-TALP). The oocytes were inseminated in IVF drops (50 #1) with approximately 50 × 105 of motile spermatozoa (pool of five BVDVnegative bulls) and then incubated at 38.5 °C in 5% CO2 in air for 18 h.
2.4. Embryo culture After IVF, the oocytes were freed from cumulus cells by repeated passage through a small-bore pipet and washed twice in D-PBS medium and once in coculture medium. The co-culture medium was INRA Menezo-B2 (Bio Merieux, Paris) supplemented with 1/A BOEC, 10% estrus cow serum (free of antibodies to BVDV and free of BVDV) and 50 #g ml- ~ gentamicin. After washing, 5-10 zygotes were placed in each droplet of co-culture medium and incubated under silicone oil (360 Medical Fluid, Dow Corning, Midland, MI) at 38.5°C in 5% CO2 in air for 8 days. The cleavage rate of oocytes was determined 72 h after insemination, and was calculated as the proportion of eggs which developed to at least the two-cell stage. At the end of incubation, the numbers of embryos that developed to morula and blastocyst stages were determined by direct microscopic examination. The washing procedure used in this study refers to the transfer of oocytes/embryos by a capillary glass pipet in approximately 3/zl medium to a clean dish containing 2 ml of fresh medium, and then gently swirling the dish and embryos for a few seconds. For each transfer a new pipet was used.
2.5. Virus assay The following samples were collected for viral assay: blood serum, follicular fluid, oviductal cells, uterine wash-fluid, granulosa cells, cumulus-oocyte complexes and embryos. The embryos (in groups of 2-5) and oviductal cells were disrupted by sonication (Ultrasonic Processor, Model GE-600, Manostat, New York, NY, USA) for 40 s at 4 °C prior to being assayed. The uterine wash fluid was obtained by washing uterine horn with 20 ml of D-PBS. All samples were frozen in cryovials and stored at - 70 ° C until assayed.
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2.6. Viral detection and titration The immunoperoxidase staining technique (Afshar et al., 1991 ) was used for BVDV detection and titration of samples. Titration was done by making ten-fold dilutions (undiluted to 10-5) in Earlie's minimum essential medium (MEM (E) ) in quadruplicate. Ten microliters of each dilution was used with a suspension of bovine turbinate (BT) cells (3 × 105 m l - ) in a tissue culture microtiter plate. After incubation for 24 h, monolayer cell cultures were fixed with 20% acetone and stained by the addition of bovine anti-BVD serum followed by a horseradish peroxidase labeled rabbit antibovine IgG conjugate. Excess conjugate was then removed by washing, following which the enzyme substrate was added and permitted to incubate until the desired staining intensity was reached. Positive cells exhibit reddish brown cytoplasmic staining in response to the precipitating substrate, whereas non-reacting cells remain clear and colorless when viewed by light microscope. Proper controls were included in each test as follows: the BT were tested with bovine anti-BVD serum, the BVDV-infected BT cells were tested with both a negative serum (BVD-antibodies free) and the bovine anti-BVD serum, and the conjugate was reacted with the BVDV-infected BT cells. Titrations were calculated by the Reed and Munch method (Davis et al., 1990). When samples were found negative with the microtiter plate immunoperoxidase test a passage was made in a flask. Briefly, bovine kidney cells, second to fifth passage, were grown in a 25 ml Falcon flask (Becton Dickinson, Lincoln Park, NJ ) in MEM (E), supplemented with 10% FCS and antibiotics. The cells were monitored routinely for BVDV; the FCS was pretested for BVDV and anti-BVDV antibody and treated with 1 Mrad of gamma irradiation at 0-4 ° C. The monolayers were washed with MEM(E), and the flask was inoculated with the sample (500/tl) to be tested. After an absorption period of 1 h at 37°C on a rocker, the cultures were maintained in MEM(E) containing 2% FCS for 5 days at 37°C. Cell cultures inoculated with BVDV samples were tested for virus antigens on Day 4 after inoculation using the immunoperoxidase test as described above.
2. 7. Statistical method The proportion of fertilized ova that cleaved, and the proportion of fertilized ova that developed to the blastocyst stage were evaluated by the Z2 test. Differences at a probability of less than 0.05 were considered significant.
3. Results
All BVDV-injected animals had biphasic fever (40-41.5 ° C ) on Days 3-4 and again Day 8 post inoculation. In addition, leukopenia accompanied fever on Days 3-4. BVDV was isolated from the sera of 15/16 and 4/16 animals on Day 4 and Day 8 post-injection, respectively. In total, 71 cumulus-oocyte complexes, 130 one-cell ova and 120 embryos obtained from infected heifers were tested for
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Table 1 BVD isolation from the samples of BDV-injected animals Samples
No. of positive animals
Viral trier (rage) (TCIDso ml- ~)
Follicular fluid Granulosa cells Oviductal cells Uterine wash-fluid Cumulus-oocyte complexes IVF embryos
16/ 16 16/ 16 0/16 4/16 16/16 0/16
103-107 102-106 Negative Positive- l 03 103-105 Negative
TCIDso - Medium tissue culture infectious dose.
BVDV. Results of virus isolation from BVDV-injected heifers are presented in Table 1. None of the non-injected animals showed any significant changes in rectal temperature (37.5-38.5 °C) or leukopenia in the blood. Also, all samples from control animals tested negative on virus isolation. From a total of 329 oocytes collected from infected animals, 258 were inseminated and cultured. A mean cleavage percentage of 47.6% (123/258) was obtained vs. 53.9% (62/115 ) for control non-infected oocytes (P> 0.05 ). The proportion of embryos that developed to blastocyst stages was significantly higher (P< 0.01 ) for the control group (29%, 18/62)than for the BVDV group (13.8%, 17/123).
4. Discussion
Inoculation of animals with BVDV induced an acute stage of disease similar to that described by Radostis and Littlejohns (1988), Corapi et al. (1989) and Brownlie (1990). It was clearly demonstrated in this experiment that the oocytes were exposed to a high titer of virus by contact with contaminated follicular fluid and the granulosa cells surrounding them in the ovaries. However, embryos produced by IVF from such oocytes were not associated with infectious virus. A number of manipulations (removing cumulus cells and washing) as well as prolonged in vitro culturing of embryos at 39 °C could account for virus inactivation or its removal. It was established previously that a simple washing procedure is sufficient to remove the virus if present at low titre ( 104-105 TCIDso m l - 1) from the intact zona pellucida of uterine stage embryos (Singh et al., 1982). Oviductal cell are in common use for co-culture of IVF-produced embryos, and may be a source of viral infection (Bielanski et al., 1993). It is surprising and difficult to explain why in the present experiment all the infected animals yielded negative oviductal cells but some had infected uterine flushes. Recently, it has been demonstrated that cows persistently infected with BVDV are poor embryo producers, but can yield uterine stage embryos which develop into non-infected offspring using embryo transfer (Wentink et al., 1991; Bak et al., 1992 ). Also, embryos produced by IVF with BVDV-infected semen from per-
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sistently infected bulls were not associated with the virus (Bielanski and Loewen, 1994). Collectively, the results as presented in this paper and reported by others indicate that the risk of transmission of BVDV through embryos fertilized in vitro is minimal provided the embryos are washed according to the procedures recommended by the International Embryo Transfer Society (Anonymous, 1990). The proportion of oocytes that cleaved from infected animals was only slightly lower than those obtained from noninfected animals. This proportion was, however, comparable with the proportion reported when semen from persistently infected bulls was used for IVF of 'clean' oocytes (Bielanski and Loewen, 1994). In contrast, a significant proportion of embryos obtained from infected animals failed to develop to the morula or blastocyst stage. The reason for this is not clearly understood because the virus is not cytopathic for early embryos in vitro (Bielanski and Hare, 1988 ). It is possible that the fever associated with the viremic stage in heifers affected the developmental competence of oocytes, as was suggested previously when oocytes from BHV-1 infected animals were fertilized in vitro (Bielanski and Dubuc, 1993). This theory might also be supported by the reported decresed fertilization and conception rates after experimental infection of heifers with BVDV around the time of insemination (McGowan et al., 1993). Overall, the results of this study indicate that low numbers of viable and transferable-stage embryos may be produced in vitro from BVDV-infected heifers and that such embryos are not associated with the virus. However, the developmental capacity of the embryos in vivo, after transfer to recipients, is still to be determined.
Acknowledgments The technical assistance of G. Raby and P. ChatiUon is gratefully acknowledged.
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