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Noncytopathic bovine viral diarrhea virus in a system for in vitro production of bovine embryos
Theriogenology
41:841-853,1994
NONCYTOPATHIC BOVINE VIRAL DIARRHEA VIRUS IN A SYSTEM FOR IN VITRO PRODUCTION OF BOVINE EMBRYOS O.V.
Zurovac,l*a D.A. Stringfellow,Lb K.V. Brock,2 M.G. Riddell and J.C. Wright'
'Department of Pathobiology and 3Department of Large Animal Surqerv _ _ and Medicine. Collece of Veterinarv Medicine Auburn University; AL 36849-55192Food Animal Health Research Program Ohio Agricultural Research and Development Center Wooster, OH 44691 USA Received
for publication: Accepted:
May 28, 1993 November 16, 1993
ABSTRACT Techniques for in vitro production of bovine embryos have evolved to the extent that applications for the commercial production of calves have been proposed. However, little is known about the epidemiological implications of the procedures. One concern is the introduction of noncytopathic bovine viral diarrhea virus (BVDV). In this study, follicular oocytes (n=247) collected from 10 cows were matured and fertilized in vitro and presumptive zygotes were cultured for 7 d. Primary cultures of bovine oviductal epithelial cells for use during in vitro fertilization and culture were divided into 2 groups. Treated oviductal cells were infected with BVDV while control cells were not exposed to the virus. Two approximately equal groups of mature oocytes from each cow were inseminated, and the presumptive zygotes were cultured with infected or noninfected oviductal cells. After 7 d in culture, zona pellucida-intact morulae/blastocysts and degenerated ova were washed, sonicated and assayed for the presence of virus. The rates of cleavage and development were also compared by Chi-square analysis. After washing, virus was not isolated from morulae and blastocysts but was isolated from some groups of degenerated ova. Infections of oviductal cells were inapparent and did not significantly (P>O.O5) affect rates of cleavage or development. Key words:
in vitro
fertilization,
bovine viral diarrhea virus
Acknowledgments The authors wish to thank Dr. Harish Minocha, Kansas State University, for supplying the anti-bovine viral diarrhea virus monoclonal antibody used in this study. aThis study is a partial fulfillment of the requirements for the degree of Master of Science. bCorrespondence and reprint requests.
Copyright
0 1994 Butterworth-Heinemann
842
Theriogenology
INTRODUCTION The latest reproductive technique that has evolved to a state of usefulness to the cattle industry is the in vitro production of embryos. In the 1970's, techniques for collection, storage, and transfer of in vivo produced bovine embryos developed to a level of usefulness before the epidemiological implications of the procedures could be properly evaluated. Thus, application of the techniques (especially in international trade) was hindered. Likewise, knowledge that allows the in vitro production and transfer of bovine embryos has superseded our knowledge of the epidemiological implications. Preimplantation-stage embryos are surrounded by a glycoproteinaceous structure called the zona pellucida (17). The zona pellucida of in vivo produced bovine embryos has been shown to serve as a barrier to penetration of pathogens (16). However, the structure of the zona pellucida of in vitro produced embryos differs (6,12), and its effectiveness as a barrier to infectious agents remains largely untested. Bovine viral diarrhea virus (BVDV) is a common pathogen among cattle, causing a variety of problems, including clinical entities such as abortion, diarrhea, and mucosal disease as well as immunotolerance and persistent infection (4). Studies using in vivo derived zona pellucida-intact bovine embryos have shown that cytopathic strains of BVDV do not penetrate the zona pellucida and do not remain adherent to the structure after proper washing (3,15). However, exposure of zona pellucida-free embryos to cytopathic and noncytopathic BVDV resulted in embryonic death and inapparent infections, respectively (8). Somatic cells, employed during in vitro fertilization and in vitro culture of presumptive zygotes to improve fertilization and development rates are a potential hazard in the system, because Recently, it was they could serve as sources of pathogens. demonstrated that granulosa cells and bovine oviductal epithelial cells could be infected inapparently with noncytopathic BVDV, thus creating a source of the virus in systems for in Vitro production of embryos (7). In addition, estrous cow serum or fetal bovine serum could serve as sources of virus for infection of somatic cell cultures (e.g., oviductal epithelial cells) prior to their use during in vitro fertilization and culture (13). In this study, we used oviductal epithelial cells that were infected with noncytopathic BVDV in duplicating procedures for in vitro fertilization and culture that are common in several laboratories including our own. A primary objective was to determine if normal-appearing embryos could be produced in the A second presence of infected oviductal epithelial cells. objective was to determine if the virus remains associated with morula and blastocysts or degenerate ova after using techniques that have been recommended for washing in vivo produced embryos (2) -
843
Theriogenology
MATERIALS Oocyte
Collection
AND METHODS
and In Vitro Maturation
Cumulus-oocyte-complexes were obtained from ten mature cows (ages 3 to 12 yr) from experimental dairy and beef production Cows were to be units within the Auburn University system. culled for a variety of reasons including age, physical Ovaries and oviducts were condition, and reproductive failure. removed surgically from donor animals and transported to the laboratory within 10 min in preheated (39OC) 0.85% saline with 2% fetal bovine serum and antibiotics (100 U penicillin G and 100 pg The cumulus-oocyte-complexes were released by streptomycin/ml). slicing the cortex of the ovaries, and were washed from the sliced surface of ovaries into a lOOO-ml glass beaker with Ham's FlO nutrient medium supplemented with 2% estrous cow serum and containing antibiotics (100 U penicillin G and 100 pg The sediment was streptomycin/ml) and 2.0 U of heparin/ml. washed 3 times with the same medium, allowing the cumulus-oocytecomplexes and other debris to settle, followed by removal of the The highest supernatants and resuspension in fresh medium. quality cumulus-oocyte-complexes (14) were retrieved with the aid of a stereomicroscope and washed three additional times in in vitro maturation medium consisting of TCM 199 with Hanks' salts supplemented with 20% estrous cow serum, 2.5 mM sodium pyruvate, and antibiotics (50 U penicillin G and 50 I.rgstreptomycin/ml). Cumulus-oocyte-complexes were matured in 50-~1 drops of the maturation medium for 24 to 26 h at 39'C in a highly humidified atmosphere of 5% CO2 and air. All microdrops for in vitro maturation (and subsequently for in vitro fertilization and culture) were overlayed with silicone oil. Collection
and culture of Oviductal
Epithelial
Cells
The oviduct ipsilateral to the ovary from which the most recent ovulation occurred was selected from each cow for preparation of primary oviductal epithelial cell cultures. Sheets of epithelial cells were stripped from oviducts by drawing the oviducts through a thin gap created by gently squeezing thumb forceps. Sheets were fragmented by sequential aspiration and discharge through a 23-gauge and then a 25-gauge needle. Fragments of oviductal epithelial cells were washed 4 times through a sequence of sedimentation, aspiration of supernatant, and resuspension in fresh in vitro culture medium. The in vitro culture medium was TCM 199 with Earle's salts supplemented with 10% estrous cow serum, 2.5 mM sodium pyruvate and antibiotics (50 U penicillin G and 50 pg streptomycin/ml). After washing, primary cultures of oviductal epithelial cells were incubated in the in vitro culture medium for 24 or 48 h before use in in vitro fertilization and in vitro culture, respectively. Prior to use in in vitro fertilization and in vitro culture, oviductal epithelial cells were washed 3 times in either HEPES-TALP (18)
844
Theriogenology
[modified Tyrodels medium containing bovine serum albumin, sodium lactate, sodium pyruvate, and gentamicin] or in vitro culture medium, respectively. In Vitro Fertilization Straws of frozen semen from the same collection of a bull of known fertility were used in all trials. The collection was determined previously to be free of BVDV by viral isolation procedures. A modification of the procedure developed by Parrish et al. was used for sperm capacitation (10). A swim-up procedure was performed in four polypropylene snap-cap tubes (12 X 75 mm), each containing 1 ml sperm-TALP (18) (modified Tyrodes's medium containing bovine serum albumin, sodium lactate, sodium pyruvate, and gentamicin). The in vitro fertilization medium consisted of fertilization-TALP (18) (modified Tyrode's medium with bovine serum albumin, sodium lactate, sodium pyruvate, gentamicin, and heparin for sperm capacitation). In preparation for in vitro fertilization, matured cumulus-oocyte-complexes with expanded cumulus were pre-washed three times in HEPES-TALP and denuded by vertical agitation as follows. Cumulus-oocyte-complexes were transferred to a well of a 4-well plate (1 ml of HEPES-TALP per well) and repeatedly aspirated and discharged through a small The procedure was bore glass pipet to remove cumulus cells. repeated in three of the wells and then oocytes were assembled in the 4th well before placing in microdrops. Prior to the addition of sperm, oocytes were transferred to lOO-~1 drops of fertilization-TALP containing oviductal epithelial cells. The oviductal epithelial cells had been washed as described and added 30 to 60 min previously. Aliquots of pelleted spermatozoa were added to the fertilization drops (a proximately 100 @) for a oocytes total concentration of about 5 X 10Y sperm cells/ml. were exposed to sperm for 16 to 18 h at 39'C in a highly humidified atmosphere of 5% CO.2and air. In Vitro Culture At the end of the fertilization period, cultures were examined for the presence of hyperactivated sperm, and presumptive zygotes were removed and prewashed in in vitro culture medium. Then, excess sperm were removed by vertical agitation (as previously described) 3 times in in vitro culture medium. Subsequently, they were transferred to in vitro culture drops (approximately 100 ~1) containing oviductal epithelial The oviductal epithelial cells had been washed as cells. described and added 30 to 60 min before presumptive zygotes were added. These presumptive zygotes were cultured for 7 d at 39'C in a humidified atmosphere of air and 5% C02. Additional in vitro culture medium was added to each drop at 48 h (50 /.~l)and 6 Cleavage at 48 h and development at 6 and 7 d were d (25 ~1). After 7 d in culture, the final number of observed. morulae/blastocysts and degenerated ova were recorded.
Theriogenology
Estrous
Cow Serum
Estrous cow serum used in in vitro maturation and in vitro culture medium was from a single lot prepared commercially (Cocalico Biologicals, Inc., Reamstown, PA 17567). The virus (noncytopathic strain SD-l) neutralizing titer of anti-BVDV antibody in this lot was 1:32. Estrous cow serum used for establishing primary oviductal epithelial cell cultures was custom prepared in our laboratory (passed through a 0.22-pm filter twice and heat inactivated at 56'C for 30 min) and failed to neutralize BVDV at a serum dilution of 1:2. Embryo/Ova
Washing
One milliliter of minimum essential medium supplemented with 10% equine serum and antibiotics (100 U penicillin G, 100 pg streptomycin, and 0.25 pg amphotericin B) in each of 12 wells of a 24-well cell culture plate constituted the washes for morulae and blastocysts or degenerated ova. Degenerated ova (groups of 10 or fewer) or morulae and blastocysts (groups of 3 or fewer) were washed by employing separate sterile micropipettes with the ova/embryo containing portion of medium carried in the pipets between washes never exceeding 10 ~1. Source and Propagation
of Virus
The virus used in this study was a noncytopathic biotype of BVDV (SD-l) that was isolated initially from the serum of a persistently infected cow at the Ohio Agricultural Research and Development Center at Wooster, Ohio. Virus was propagated in BVDV-free Madin-Darby bovine kidney (MDBK) cells. Cells were grown in minimum essential medium with Earle's salts supplemented with 10% equine serum and antibiotics (100 U penicillin G, 100 pg streptomycin, and 0.25 pg amphotericin B). Nearly confluent monolayers were inoculated with virus received from the Ohio Agricultural Research and Development Center and incubated for 72 h at 37OC in a humidified atmosphere of 5% CO2 and air. Stock virus was harvested by a single freeze-and-thaw method, aliquoted and frozen at -6O'C until used. The TCIDSO of stock virus (lXlO'/ml) was determined prior to the beginning of experiments. Virus Detection Virus was isolated in BVDV-free MDBK cells that were cultured in minimum essential medium as described for 72 h after exposure to the test samples. The method for detection of the noncytopathic BVDV was reported previously (1). Briefly, the procedure involved incubation of exposed cells with anti-BVDV monoclonal antibody, washing to remove unbound antibody, addition of peroxidase conjugated rabbit anti-mouse IgG, washing to remove
Theriogenology
846
unbound conjugated antibody, and finally, the addition of aminoethyl carbazole which produces reddish-brown granules when oxidized by horseradish peroxidase. The color change was detected using a light microscope and evaluated with the benefit of comparison to known infected and uninfected controls. Virus
Isolation
A summary of samples from which virus isolation was attempted and the sequence in which they were assayed is presented in Table 1. Due to the small amounts of material available for assay, specimens from in vitro fertilization and in vitro culture drops were assayed directly in 96-well cell culture plates as follows: Oviductal epithelial cells and associated media were placed in a microcentrifuge tube and centrifuged at 15,600 X G for 5 min. Supernatants were assayed immediately, but pelleted oviductal cells were frozen (-60°C) and thawed once to enable viral release prior to assay. Then 3 replicate wells containing 99 pl of minimum essential medium (supplemented as described) were inoculated with ll-).~laliguots of BVDV-exposed or control sample. Each sample was serially diluted (10' to 10') and overlaid with 26 1.11of MDBK cells suspended in the culture medium. Virus-free negative controls also were propagated in 3 replicates while titration of the stock virus served as a positive control. After inoculation of test and control samples, plates were incubated for 72 h at 37'C in a humidified atmosphere of 5% CO2 and air before the immunoperoxidase labeling was applied. Table 1. Assayed a) b) Assayed :; e) f) g)
Samples assayed for the presence bovine viral diarrhea virus after the fertilization
period
of noncytopathic
(16 to 18 hours):
Oviductal cells from fertilization Medium from fertilization drops after the in vitro culture period
drops
(7 days):
Oviductal cells from in vitro culture drops Medium from in vitro culture drops Washed and sonicated groups of degenerate ova Washed and sonicated morulae and blastocysts Last three washes for each group of embryos or degenerated ova
Note: virus isolation was attempted well as from virus exposed samples. Morulae/blastocysts described and sonicated
from control
(nonexposed) as
and degenerated ova were washed as (2 bursts at 35% for 60 set each time
847
Theriogenology
with a Model 300 Artek Sonic Dismembrator, Fisher Scientific, Co, Pittsburgh, PA 15219). Sonicate fluids were inoculated onto MDBK cells and passaged three times (5-d incubation per passage) to provide maximum opportunity for viral replication before virus was released by a single freeze-and-thaw method, and aliquots were assayed for virus in 96-well plates as described. The last 3 washes for each group of morulae/blastocysts or degenerated ova were overlaid with MDBK cells and incubated for 5 d as described. Then cells were disrupted by a single freezeand-thaw method, and aliquots from each wash culture were assayed for virus in a 96-well plate as described. Experimental
Design
For each cow, primary cultures of oviductal epithelial cells were established in 4-well cell culture plates. Two cultures of oviductal cells were exposed to noncytopathic BVDV while the other two were not exposed. One set of exposed and nonexposed oviductal cell cultures were incubated for 24 h and used in the fertilization drops, while the other set was incubated for 48 h Cumulus-oocyte-complexes and used in the in vitro culture drops. from each cow also were divided into approximately equal groups. After in vitro maturation of the oocytes, treatment groups were co-cultured with virus-exposed oviductal epithelial cells, while the controls were co-cultured with nonexposed oviductal epithelial cells during both in vitro fertilization and in vitro culture. At Day 7 after the beginning of in vitro culture, zona pellucida-intact morulae and blastocysts or zona pellucida-intact degenerated ova were washed, sonicated and assayed for virus as The last 3 washes for each group of degenerated ova described. or morulae and blastocysts also were assayed for presence of virus to confirm the efficacy of the washing procedure for removing free virus. After presumptive zygotes were removed at the end of the in vitro fertilization period (16 to 18 h) and degenerated ova and morulae/blastocysts were removed at the end of the in vitro culture period (7 d), viral presence or absence (in the fertilization or culture drops) was determined as described. All samples described above from both treated and control groups were assayed for the presence of virus. Differences in rates of cleavage and development between treatment and control groups were compared by Chi-square analysis (19). RESULTS A total of 247 cumulus-oocyte-complexes of acceptable quality were recovered from the 10 cows and assigned to treatment A comparison of cleavage (n = 124) and control (n = 123) groups. and development rates is presented in Table 2. The rates of cleavage and development were not statistically different between treatment and control groups (P > 0.05, Chi-square test for independence).
848
Theriogenology
Bovine viral diarrhea virus was isolated from the oviductal epithelial cells collected from fertilization drops after the period of fertilization and from the oviductal epithelial cells collected from in vitro culture drops after 7 d of culture for all treatment groups, while the virus was not isolated from any of the same samples from control groups. Regarding morphology and ciliary action, light microscopic examinations of the cells revealed no observable differences between infected and noninfected oviductal epithelial cells. Further, hyperactive sperm always were observed at the end of the fertilization period, and no difference was observed between drops with or without virus. A summary of virus isolations for samples assayed from treated groups is presented in Table 3. Although BVDV had been isolated from oviductal epithelial cells in the in vitro fertilization and in vitro culture drops of all treatment groups, the virus was not isolated from any of the corresponding in vitro culture medium and was only isolated from the corresponding medium from the fertilization drops for 4 of the 10 cows. Although BVDV was not isolated from any of the last three washes for any treated group of degenerate ova, the virus was recovered from the sonicate fluids of 2 of the 15 groups assayed. The virus was not isolated from any washes or sonicate fluids of treated morulae and blastocysts. Finally, there was no observable difference between morulae and blastocysts produced in the presence or absence of BVDV-infected oviductal epithelial cells.
Table 2.
Number of cumulus-oocyte-complexes and rates of cleavage (at 48 hours) and development (at 7 days) of presumptive zygotes produced by fertilization and culture in the presence of oviductal epithelial cells infected (treated) or not infected (control) with noncytopathic bovine viral diarrhea virus P
Number of cumulusoocyte-complexes
Control
Treated
123
124
Number of cleaved zygotes (%)
0.31
25
(23%)
32
(28%)
Number morulae/ blastocysts (S)
0.39
8
(32%)
7
(22%)
Note: estrous cow serum in the in vitro culture medium had antibovine viral diarrhea virus antibody (virus neutralizing titer = P = probability of a larger Chi-square. 1:32).
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Theriogenology
Table
3.
Isolation of bovine viral diarrhea virus from samples associated with in vitro fertilization (IVF) and in vitro culture (IVC) in the presence of bovine oviductal epithelial cells (BOEC) infected with the virus
Sample
No. positive/Total
BOEC from IVF drops Medium
from IVF drops
BOEC from IVC drops Medium
from IVC drops
Groups of degenerated
ova
Morulae/blastocysts Note: virus was not isolated
no.
(% Positive)
lO/lO
100%
4/10
40%
IO/10
100%
O/IO
0%
2/15
13%
U/7
0%
from any control samples. DISCUSSION
The potential for exposure of transfer-stage embryos to BVDV has been a subject of several reports. Bielanski and Hare exposed in vivo produced Day-7 embryos (with the zona pellucida intact, damaged, or removed) to a cytopathic strain of BVDV (5). No effect on embryonic survival or development was observed after 24 or 48 h in culture. However, cytopathic effects might have been observed if the culture time had been extended. A similar study conducted by Potter et al., also using in vivo-derived embryos, resulted in failure to isolate BVDV from sonicated embryos that had been exposed to the virus for 24 h and washed 4 times (11). Other studies in which Day-7 zona pellucida-intact embryos were collected from superovulated cows, exposed to cytopathic BVDV, and assayed for virus resulted in isolation of the virus from embryos when washing was inadequate (9), but failure to isolate the virus when washing was thorough (15). Thus, the intactness of the zona pellucida of in vivo-derived bovine embryos appears to provide a barrier to penetration or firm attachment by BVDV. In a recent study, Day-7 embryos were collected from superovulated cows, hatched in vitro, exposed to cytopathic or noncytopathic strains of BVDV, and subsequently cultured for up to 14 d (8). Cytopathic effect was observed in those that were exposed to cytopathic virus, but those that were exposed to noncytopathic BVDV expanded normally when compared to nonexposed controls. Infection with noncytopathic BVDV was confirmed in the apparently normal, developing embryos at the end of in vitro culture (8). Thus, zona pellucida-free in vivo-derived bovine
Theriogenology
embryos were susceptible to infection with BVDV, and infection with the noncytopathic virus did not have an apparent effect on development. Systems for in vitro production of bovine embryos do provide the opportunity for close and continuous observation of oocytes, the conceptus, and support cells; therefore, infectious agents causing observable abnormalities in support cells could be detected, and agents providing a total block to fertilization or development would not constitute an epidemiologic hazard since no embryos would be produced for transfer. However, infectious agents that do not cause observable cytopathic effects or do not dramatically reduce fertilization or embryonic development could easily escape detection and are of serious concern. In our earlier study, zona pellucida-free bovine embryos continued to develop without observable changes after infection with noncytopathic biotypes of BVDV (8). Preliminary data (unpublished) indicated that the SD-l isolate of noncytopathic BVDV would cause infections of bovine oviductal epithelial cells that were inapparent after short-term in vitro culture. Thus, the SD-l isolate was selected for use in this study. Isolation of the noncytopathic BVDV from all exposed oviductal epithelial cells confirmed that the virus was in the system during the time of fertilization and throughout the period of in vitro culture for treated groups of oocytes and presumptive zygotes/embryos, respectively. Failure to isolate the virus from all unexposed oviductal epithelial cells, provided reassurance that the virus had not been introduced inadvertently (via reagents or tissues) into the in vitro fertilization or in vitro culture drops of control oocytes and presumptive zygotes. All commercial lots of estrous cow serum that we had tested contained antibody to BVDV. Therefore, we elected to use estrous cow serum with anti-BVDV antibody in the in vitro culture medium, since this was representative of the type of serum likely to be used routinely in in vitro fertilization in our laboratory as well as in some other laboratories. Because specific antibody was present, it is not surprising that the virus never was isolated from medium collected from in vitro culture drops despite the presence of infected oviductal epithelial cells in all of these drops. However, it was initially surprising that we were only able to isolate the virus from the fertilization-TALP from 60% of the fertilization drops when infected oviductal epithelial cells were present in all of these drops. This finding is indicative of the presence of a component (or multiple components) in the in vitro fertilization medium with an inhibitory effect on the infectivity of the virus. Results of our additional preliminary study (not included) showed that the presence of bovine serum albumin was associated with reduced levels of free infectious BVDV in the fertilization-TALP. Although we have no proof, it is possible that immunoglobulin contamination of the bovine serum albumin was responsible for this anti-BVDV activity.
Theriogenology
851
Obviously, the anti-BVDV antibody in the in vitro culture medium and the undefined anti-BVDV activity in the fertilizationTALP, limited the exposure of oocytes and zygotes to free infectious virus during in vitro fertilization and in vitro However, these obstacles did not provide culture, respectively. total protection, since the virus was isolated from two groups of The mechanism by which the BVDV degenerated ova after washing. Virus was associated with these degenerated ova is uncertain. might have been carried across the zona pellucida at the time of fertilization or it could have become attached to or penetrated the zona pellucida at any time during the period of in vitro Regardless, it is important to note that these virusculture. positive ova were zona pellucida-intact and washed according to procedures that would have been effective for insuring that in vivo-derived zona pellucida-intact embryos were free of BVDV. Certainly, there is a need to wash in vitro-derived embryos: however, in light of our findings, it appears that additional precautions might have to be taken to insure freedom of these embryos from BVDV. The cleavage and development rates for both treated and control groups were slightly lower than expected from similar This may applications of these techniques in our laboratory. have been due to the additional time outside of the environment of the incubator that was required for separate handling of the treatment groups. Regardless, rates of cleavage and development were sufficient to allow statistical and visual comparisons that indicated no difference between groups of oocytes and presumptive zygotes fertilized and cultured in the presence or absence of oviductal epithelial cells that were infected with noncytopathic BVDV. It was not surprising to us that the presence of noncytopathic BVDV in oviductal epithelial cells was not visually apparent and did not result in differences in rates of cleavage and development; however, these results do raise some concern. As stated previously, key animal tissues (e.g., oocytes, cumulus cells, and oviductal epithelial cells) or sera used in the in vitro production of bovine embryos provide the potential for introduction of BVDV (7,13), yet guidelines for quality control of these components of in vitro fertilization systems have not been established. It was encouraging that no virus was isolated from morulae or blastocysts (that were of transferrable quality) after washing, and it is tempting to speculate that the isolations from degenerate ova may have been the result of "lethal infections IIthat do not constitute an epidemiological hazard since these embryos are not available for transfer. However, the number of viable embryos that were assayed was small, and‘it is possible that testing of additional morulae and blastocysts might have resulted in some positives. In this study, noncytopathic BVDV was introduced into a representative system for in vitro production of bovine embryos in a preliminary attempt to investigate epidemiological hazards
Theriogenology
852
associated with the virus. Infections of oviductal epithelial cells were not readily apparent and did not affect rates of cleavage and development or the apparent quality of morulae and blastocysts that were produced: therefore, there is a need for specific quality control measures to insure that systems for in vitro production of bovine embryos do not contain noncytopathic BVDV. Based on the current study, the.efficacy of using washing procedures recommended for insuring that in vivo-derived bovine embryos are free of BVDV to insure that in vitro-derived embryos are free of the virus has to be questioned. Additional work will be required to properly evaluate washing procedures for in vitroderived embryos.
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Afshar A, Dulac GC, Dubuc C, Howard TH. 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;55:91-93. Anonymous. Recommendations for the sanitary handling of embryos. In: Stringfellow DA, Seidel SM (eds), Manual of the International Embryo Transfer Society. Second edition. IETS, Champaign, IL, 1990;41-45. Archbald LF, Fulton RW, Seger CL, Al-Bagdadi F, Godke RA. Effect of the bovine viral diarrhea (BVD) virus on preimplantation bovine embryos: a preliminary study. Theriogenology 1979;11:80-89. Baker JC. Bovine viral diarrhea virus: a review. J Am Vet Med Assoc 1987;190:1449-1458. Bielanski A, Hare WCD. Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact, damaged and removed. Vet Res Corn 1988;12:19-24. Boice ML, Mavrogianis PA, Murphy CN, Prather RS, Day BN. Immunocytochemical analysis of the association of bovine oviduct-specific glycoproteins with early embryos. J Exp 2001 1992;263:225-229. Booth PJ, Stevens DA, Collins ME, Brownlie J. Detection of bovine viral diarrhea virus (BVDV) in ovarian and oviductal tissue. J Reprod Fertil Abs Ser 1992;9:28. Brock EV, Stringfellow Comparative effects of cytopathic and DA. noncytopathic bovine viral diarrhea virus on bovine blastocysts. Theriogenology 1993;39:196 abstr. Gillespie JH, Schlafer DH, Foote RH, Quick S, Dougherty E, Schiff E. Allen S. Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure and washing. Dtsch Tierarztl Wschr 1990;97:77-87. JL, Leibfried-Rutledge ML, Crister ES, Parrish JJ, Susko-Parrish Eyestone WH, First NL. Bovine in vitro fertilization with frozenthawed semen. Theriogenology 1986;25:591-600. Potter ML, Corstvet RE, Looney CR, Fulton RW, Archbald LF, Godke RA. Evaluation of bovine viral diarrhea virus uptake by preimplantation embryos. Am J Vet Res 1984;45:1778-1780. Monoclonal Riddell KP, Stringfellow DA, Panangala VS, Scodras JM. antibodies to the bovine zona pellucida. Theriogenology 1992;37:285 abstr.
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Viral contamination of bovine Rossi CR, Bridgeman CR, Kiesel GK. Am J Vet Res 1980; fetal lung cultures and bovine fetal serum. 41:1680-1681. In vitro Shioya Y, Kuwayama M, Fukushima M, Iwasaki S, Banada A. fertilization and cleavage capability of bovine follicular oocytes Theriogenology classified by cumulus cells and matured in vitro. 1988;30:489-496. Embryo Singh EL, Eaglesome MD, Thomas FC, Papp-Vid G! Bare WCD. transfer as a means of controlling the transmission of viral infections. I. The in vitro exposure of preimplantation bovine embryos to akabane, bluetongue, and bovine viral diarrhea viruses. Theriogenology 1982;17:437-444. The potential of embryo Stringfellow DA, Riddell KP, Zurovac 0. NZ Vet J 1991; transfer for infectious disease control in livestock. 39:8-17. Ann Rev Biochem 1988; Wassarman PM. Zona pellucida glycoproteins. 57~415-442. Pregnancy resulting from cattle Xu KP, Greve T, Callesen H, Hyttel P. J Reprod Fertil 1987;81:501oocytes matured and fertilized in vitro. 504. Analysis. Second edition, Prentice-Hall, Inc., Zar JH. Biostatistical Englewood Cliffs, New Jersey, 1984;48.
41:841-853,1994
NONCYTOPATHIC BOVINE VIRAL DIARRHEA VIRUS IN A SYSTEM FOR IN VITRO PRODUCTION OF BOVINE EMBRYOS O.V.
Zurovac,l*a D.A. Stringfellow,Lb K.V. Brock,2 M.G. Riddell and J.C. Wright'
'Department of Pathobiology and 3Department of Large Animal Surqerv _ _ and Medicine. Collece of Veterinarv Medicine Auburn University; AL 36849-55192Food Animal Health Research Program Ohio Agricultural Research and Development Center Wooster, OH 44691 USA Received
for publication: Accepted:
May 28, 1993 November 16, 1993
ABSTRACT Techniques for in vitro production of bovine embryos have evolved to the extent that applications for the commercial production of calves have been proposed. However, little is known about the epidemiological implications of the procedures. One concern is the introduction of noncytopathic bovine viral diarrhea virus (BVDV). In this study, follicular oocytes (n=247) collected from 10 cows were matured and fertilized in vitro and presumptive zygotes were cultured for 7 d. Primary cultures of bovine oviductal epithelial cells for use during in vitro fertilization and culture were divided into 2 groups. Treated oviductal cells were infected with BVDV while control cells were not exposed to the virus. Two approximately equal groups of mature oocytes from each cow were inseminated, and the presumptive zygotes were cultured with infected or noninfected oviductal cells. After 7 d in culture, zona pellucida-intact morulae/blastocysts and degenerated ova were washed, sonicated and assayed for the presence of virus. The rates of cleavage and development were also compared by Chi-square analysis. After washing, virus was not isolated from morulae and blastocysts but was isolated from some groups of degenerated ova. Infections of oviductal cells were inapparent and did not significantly (P>O.O5) affect rates of cleavage or development. Key words:
in vitro
fertilization,
bovine viral diarrhea virus
Acknowledgments The authors wish to thank Dr. Harish Minocha, Kansas State University, for supplying the anti-bovine viral diarrhea virus monoclonal antibody used in this study. aThis study is a partial fulfillment of the requirements for the degree of Master of Science. bCorrespondence and reprint requests.
Copyright
0 1994 Butterworth-Heinemann
842
Theriogenology
INTRODUCTION The latest reproductive technique that has evolved to a state of usefulness to the cattle industry is the in vitro production of embryos. In the 1970's, techniques for collection, storage, and transfer of in vivo produced bovine embryos developed to a level of usefulness before the epidemiological implications of the procedures could be properly evaluated. Thus, application of the techniques (especially in international trade) was hindered. Likewise, knowledge that allows the in vitro production and transfer of bovine embryos has superseded our knowledge of the epidemiological implications. Preimplantation-stage embryos are surrounded by a glycoproteinaceous structure called the zona pellucida (17). The zona pellucida of in vivo produced bovine embryos has been shown to serve as a barrier to penetration of pathogens (16). However, the structure of the zona pellucida of in vitro produced embryos differs (6,12), and its effectiveness as a barrier to infectious agents remains largely untested. Bovine viral diarrhea virus (BVDV) is a common pathogen among cattle, causing a variety of problems, including clinical entities such as abortion, diarrhea, and mucosal disease as well as immunotolerance and persistent infection (4). Studies using in vivo derived zona pellucida-intact bovine embryos have shown that cytopathic strains of BVDV do not penetrate the zona pellucida and do not remain adherent to the structure after proper washing (3,15). However, exposure of zona pellucida-free embryos to cytopathic and noncytopathic BVDV resulted in embryonic death and inapparent infections, respectively (8). Somatic cells, employed during in vitro fertilization and in vitro culture of presumptive zygotes to improve fertilization and development rates are a potential hazard in the system, because Recently, it was they could serve as sources of pathogens. demonstrated that granulosa cells and bovine oviductal epithelial cells could be infected inapparently with noncytopathic BVDV, thus creating a source of the virus in systems for in Vitro production of embryos (7). In addition, estrous cow serum or fetal bovine serum could serve as sources of virus for infection of somatic cell cultures (e.g., oviductal epithelial cells) prior to their use during in vitro fertilization and culture (13). In this study, we used oviductal epithelial cells that were infected with noncytopathic BVDV in duplicating procedures for in vitro fertilization and culture that are common in several laboratories including our own. A primary objective was to determine if normal-appearing embryos could be produced in the A second presence of infected oviductal epithelial cells. objective was to determine if the virus remains associated with morula and blastocysts or degenerate ova after using techniques that have been recommended for washing in vivo produced embryos (2) -
843
Theriogenology
MATERIALS Oocyte
Collection
AND METHODS
and In Vitro Maturation
Cumulus-oocyte-complexes were obtained from ten mature cows (ages 3 to 12 yr) from experimental dairy and beef production Cows were to be units within the Auburn University system. culled for a variety of reasons including age, physical Ovaries and oviducts were condition, and reproductive failure. removed surgically from donor animals and transported to the laboratory within 10 min in preheated (39OC) 0.85% saline with 2% fetal bovine serum and antibiotics (100 U penicillin G and 100 pg The cumulus-oocyte-complexes were released by streptomycin/ml). slicing the cortex of the ovaries, and were washed from the sliced surface of ovaries into a lOOO-ml glass beaker with Ham's FlO nutrient medium supplemented with 2% estrous cow serum and containing antibiotics (100 U penicillin G and 100 pg The sediment was streptomycin/ml) and 2.0 U of heparin/ml. washed 3 times with the same medium, allowing the cumulus-oocytecomplexes and other debris to settle, followed by removal of the The highest supernatants and resuspension in fresh medium. quality cumulus-oocyte-complexes (14) were retrieved with the aid of a stereomicroscope and washed three additional times in in vitro maturation medium consisting of TCM 199 with Hanks' salts supplemented with 20% estrous cow serum, 2.5 mM sodium pyruvate, and antibiotics (50 U penicillin G and 50 I.rgstreptomycin/ml). Cumulus-oocyte-complexes were matured in 50-~1 drops of the maturation medium for 24 to 26 h at 39'C in a highly humidified atmosphere of 5% CO2 and air. All microdrops for in vitro maturation (and subsequently for in vitro fertilization and culture) were overlayed with silicone oil. Collection
and culture of Oviductal
Epithelial
Cells
The oviduct ipsilateral to the ovary from which the most recent ovulation occurred was selected from each cow for preparation of primary oviductal epithelial cell cultures. Sheets of epithelial cells were stripped from oviducts by drawing the oviducts through a thin gap created by gently squeezing thumb forceps. Sheets were fragmented by sequential aspiration and discharge through a 23-gauge and then a 25-gauge needle. Fragments of oviductal epithelial cells were washed 4 times through a sequence of sedimentation, aspiration of supernatant, and resuspension in fresh in vitro culture medium. The in vitro culture medium was TCM 199 with Earle's salts supplemented with 10% estrous cow serum, 2.5 mM sodium pyruvate and antibiotics (50 U penicillin G and 50 pg streptomycin/ml). After washing, primary cultures of oviductal epithelial cells were incubated in the in vitro culture medium for 24 or 48 h before use in in vitro fertilization and in vitro culture, respectively. Prior to use in in vitro fertilization and in vitro culture, oviductal epithelial cells were washed 3 times in either HEPES-TALP (18)
844
Theriogenology
[modified Tyrodels medium containing bovine serum albumin, sodium lactate, sodium pyruvate, and gentamicin] or in vitro culture medium, respectively. In Vitro Fertilization Straws of frozen semen from the same collection of a bull of known fertility were used in all trials. The collection was determined previously to be free of BVDV by viral isolation procedures. A modification of the procedure developed by Parrish et al. was used for sperm capacitation (10). A swim-up procedure was performed in four polypropylene snap-cap tubes (12 X 75 mm), each containing 1 ml sperm-TALP (18) (modified Tyrodes's medium containing bovine serum albumin, sodium lactate, sodium pyruvate, and gentamicin). The in vitro fertilization medium consisted of fertilization-TALP (18) (modified Tyrode's medium with bovine serum albumin, sodium lactate, sodium pyruvate, gentamicin, and heparin for sperm capacitation). In preparation for in vitro fertilization, matured cumulus-oocyte-complexes with expanded cumulus were pre-washed three times in HEPES-TALP and denuded by vertical agitation as follows. Cumulus-oocyte-complexes were transferred to a well of a 4-well plate (1 ml of HEPES-TALP per well) and repeatedly aspirated and discharged through a small The procedure was bore glass pipet to remove cumulus cells. repeated in three of the wells and then oocytes were assembled in the 4th well before placing in microdrops. Prior to the addition of sperm, oocytes were transferred to lOO-~1 drops of fertilization-TALP containing oviductal epithelial cells. The oviductal epithelial cells had been washed as described and added 30 to 60 min previously. Aliquots of pelleted spermatozoa were added to the fertilization drops (a proximately 100 @) for a oocytes total concentration of about 5 X 10Y sperm cells/ml. were exposed to sperm for 16 to 18 h at 39'C in a highly humidified atmosphere of 5% CO.2and air. In Vitro Culture At the end of the fertilization period, cultures were examined for the presence of hyperactivated sperm, and presumptive zygotes were removed and prewashed in in vitro culture medium. Then, excess sperm were removed by vertical agitation (as previously described) 3 times in in vitro culture medium. Subsequently, they were transferred to in vitro culture drops (approximately 100 ~1) containing oviductal epithelial The oviductal epithelial cells had been washed as cells. described and added 30 to 60 min before presumptive zygotes were added. These presumptive zygotes were cultured for 7 d at 39'C in a humidified atmosphere of air and 5% C02. Additional in vitro culture medium was added to each drop at 48 h (50 /.~l)and 6 Cleavage at 48 h and development at 6 and 7 d were d (25 ~1). After 7 d in culture, the final number of observed. morulae/blastocysts and degenerated ova were recorded.
Theriogenology
Estrous
Cow Serum
Estrous cow serum used in in vitro maturation and in vitro culture medium was from a single lot prepared commercially (Cocalico Biologicals, Inc., Reamstown, PA 17567). The virus (noncytopathic strain SD-l) neutralizing titer of anti-BVDV antibody in this lot was 1:32. Estrous cow serum used for establishing primary oviductal epithelial cell cultures was custom prepared in our laboratory (passed through a 0.22-pm filter twice and heat inactivated at 56'C for 30 min) and failed to neutralize BVDV at a serum dilution of 1:2. Embryo/Ova
Washing
One milliliter of minimum essential medium supplemented with 10% equine serum and antibiotics (100 U penicillin G, 100 pg streptomycin, and 0.25 pg amphotericin B) in each of 12 wells of a 24-well cell culture plate constituted the washes for morulae and blastocysts or degenerated ova. Degenerated ova (groups of 10 or fewer) or morulae and blastocysts (groups of 3 or fewer) were washed by employing separate sterile micropipettes with the ova/embryo containing portion of medium carried in the pipets between washes never exceeding 10 ~1. Source and Propagation
of Virus
The virus used in this study was a noncytopathic biotype of BVDV (SD-l) that was isolated initially from the serum of a persistently infected cow at the Ohio Agricultural Research and Development Center at Wooster, Ohio. Virus was propagated in BVDV-free Madin-Darby bovine kidney (MDBK) cells. Cells were grown in minimum essential medium with Earle's salts supplemented with 10% equine serum and antibiotics (100 U penicillin G, 100 pg streptomycin, and 0.25 pg amphotericin B). Nearly confluent monolayers were inoculated with virus received from the Ohio Agricultural Research and Development Center and incubated for 72 h at 37OC in a humidified atmosphere of 5% CO2 and air. Stock virus was harvested by a single freeze-and-thaw method, aliquoted and frozen at -6O'C until used. The TCIDSO of stock virus (lXlO'/ml) was determined prior to the beginning of experiments. Virus Detection Virus was isolated in BVDV-free MDBK cells that were cultured in minimum essential medium as described for 72 h after exposure to the test samples. The method for detection of the noncytopathic BVDV was reported previously (1). Briefly, the procedure involved incubation of exposed cells with anti-BVDV monoclonal antibody, washing to remove unbound antibody, addition of peroxidase conjugated rabbit anti-mouse IgG, washing to remove
Theriogenology
846
unbound conjugated antibody, and finally, the addition of aminoethyl carbazole which produces reddish-brown granules when oxidized by horseradish peroxidase. The color change was detected using a light microscope and evaluated with the benefit of comparison to known infected and uninfected controls. Virus
Isolation
A summary of samples from which virus isolation was attempted and the sequence in which they were assayed is presented in Table 1. Due to the small amounts of material available for assay, specimens from in vitro fertilization and in vitro culture drops were assayed directly in 96-well cell culture plates as follows: Oviductal epithelial cells and associated media were placed in a microcentrifuge tube and centrifuged at 15,600 X G for 5 min. Supernatants were assayed immediately, but pelleted oviductal cells were frozen (-60°C) and thawed once to enable viral release prior to assay. Then 3 replicate wells containing 99 pl of minimum essential medium (supplemented as described) were inoculated with ll-).~laliguots of BVDV-exposed or control sample. Each sample was serially diluted (10' to 10') and overlaid with 26 1.11of MDBK cells suspended in the culture medium. Virus-free negative controls also were propagated in 3 replicates while titration of the stock virus served as a positive control. After inoculation of test and control samples, plates were incubated for 72 h at 37'C in a humidified atmosphere of 5% CO2 and air before the immunoperoxidase labeling was applied. Table 1. Assayed a) b) Assayed :; e) f) g)
Samples assayed for the presence bovine viral diarrhea virus after the fertilization
period
of noncytopathic
(16 to 18 hours):
Oviductal cells from fertilization Medium from fertilization drops after the in vitro culture period
drops
(7 days):
Oviductal cells from in vitro culture drops Medium from in vitro culture drops Washed and sonicated groups of degenerate ova Washed and sonicated morulae and blastocysts Last three washes for each group of embryos or degenerated ova
Note: virus isolation was attempted well as from virus exposed samples. Morulae/blastocysts described and sonicated
from control
(nonexposed) as
and degenerated ova were washed as (2 bursts at 35% for 60 set each time
847
Theriogenology
with a Model 300 Artek Sonic Dismembrator, Fisher Scientific, Co, Pittsburgh, PA 15219). Sonicate fluids were inoculated onto MDBK cells and passaged three times (5-d incubation per passage) to provide maximum opportunity for viral replication before virus was released by a single freeze-and-thaw method, and aliquots were assayed for virus in 96-well plates as described. The last 3 washes for each group of morulae/blastocysts or degenerated ova were overlaid with MDBK cells and incubated for 5 d as described. Then cells were disrupted by a single freezeand-thaw method, and aliquots from each wash culture were assayed for virus in a 96-well plate as described. Experimental
Design
For each cow, primary cultures of oviductal epithelial cells were established in 4-well cell culture plates. Two cultures of oviductal cells were exposed to noncytopathic BVDV while the other two were not exposed. One set of exposed and nonexposed oviductal cell cultures were incubated for 24 h and used in the fertilization drops, while the other set was incubated for 48 h Cumulus-oocyte-complexes and used in the in vitro culture drops. from each cow also were divided into approximately equal groups. After in vitro maturation of the oocytes, treatment groups were co-cultured with virus-exposed oviductal epithelial cells, while the controls were co-cultured with nonexposed oviductal epithelial cells during both in vitro fertilization and in vitro culture. At Day 7 after the beginning of in vitro culture, zona pellucida-intact morulae and blastocysts or zona pellucida-intact degenerated ova were washed, sonicated and assayed for virus as The last 3 washes for each group of degenerated ova described. or morulae and blastocysts also were assayed for presence of virus to confirm the efficacy of the washing procedure for removing free virus. After presumptive zygotes were removed at the end of the in vitro fertilization period (16 to 18 h) and degenerated ova and morulae/blastocysts were removed at the end of the in vitro culture period (7 d), viral presence or absence (in the fertilization or culture drops) was determined as described. All samples described above from both treated and control groups were assayed for the presence of virus. Differences in rates of cleavage and development between treatment and control groups were compared by Chi-square analysis (19). RESULTS A total of 247 cumulus-oocyte-complexes of acceptable quality were recovered from the 10 cows and assigned to treatment A comparison of cleavage (n = 124) and control (n = 123) groups. and development rates is presented in Table 2. The rates of cleavage and development were not statistically different between treatment and control groups (P > 0.05, Chi-square test for independence).
848
Theriogenology
Bovine viral diarrhea virus was isolated from the oviductal epithelial cells collected from fertilization drops after the period of fertilization and from the oviductal epithelial cells collected from in vitro culture drops after 7 d of culture for all treatment groups, while the virus was not isolated from any of the same samples from control groups. Regarding morphology and ciliary action, light microscopic examinations of the cells revealed no observable differences between infected and noninfected oviductal epithelial cells. Further, hyperactive sperm always were observed at the end of the fertilization period, and no difference was observed between drops with or without virus. A summary of virus isolations for samples assayed from treated groups is presented in Table 3. Although BVDV had been isolated from oviductal epithelial cells in the in vitro fertilization and in vitro culture drops of all treatment groups, the virus was not isolated from any of the corresponding in vitro culture medium and was only isolated from the corresponding medium from the fertilization drops for 4 of the 10 cows. Although BVDV was not isolated from any of the last three washes for any treated group of degenerate ova, the virus was recovered from the sonicate fluids of 2 of the 15 groups assayed. The virus was not isolated from any washes or sonicate fluids of treated morulae and blastocysts. Finally, there was no observable difference between morulae and blastocysts produced in the presence or absence of BVDV-infected oviductal epithelial cells.
Table 2.
Number of cumulus-oocyte-complexes and rates of cleavage (at 48 hours) and development (at 7 days) of presumptive zygotes produced by fertilization and culture in the presence of oviductal epithelial cells infected (treated) or not infected (control) with noncytopathic bovine viral diarrhea virus P
Number of cumulusoocyte-complexes
Control
Treated
123
124
Number of cleaved zygotes (%)
0.31
25
(23%)
32
(28%)
Number morulae/ blastocysts (S)
0.39
8
(32%)
7
(22%)
Note: estrous cow serum in the in vitro culture medium had antibovine viral diarrhea virus antibody (virus neutralizing titer = P = probability of a larger Chi-square. 1:32).
849
Theriogenology
Table
3.
Isolation of bovine viral diarrhea virus from samples associated with in vitro fertilization (IVF) and in vitro culture (IVC) in the presence of bovine oviductal epithelial cells (BOEC) infected with the virus
Sample
No. positive/Total
BOEC from IVF drops Medium
from IVF drops
BOEC from IVC drops Medium
from IVC drops
Groups of degenerated
ova
Morulae/blastocysts Note: virus was not isolated
no.
(% Positive)
lO/lO
100%
4/10
40%
IO/10
100%
O/IO
0%
2/15
13%
U/7
0%
from any control samples. DISCUSSION
The potential for exposure of transfer-stage embryos to BVDV has been a subject of several reports. Bielanski and Hare exposed in vivo produced Day-7 embryos (with the zona pellucida intact, damaged, or removed) to a cytopathic strain of BVDV (5). No effect on embryonic survival or development was observed after 24 or 48 h in culture. However, cytopathic effects might have been observed if the culture time had been extended. A similar study conducted by Potter et al., also using in vivo-derived embryos, resulted in failure to isolate BVDV from sonicated embryos that had been exposed to the virus for 24 h and washed 4 times (11). Other studies in which Day-7 zona pellucida-intact embryos were collected from superovulated cows, exposed to cytopathic BVDV, and assayed for virus resulted in isolation of the virus from embryos when washing was inadequate (9), but failure to isolate the virus when washing was thorough (15). Thus, the intactness of the zona pellucida of in vivo-derived bovine embryos appears to provide a barrier to penetration or firm attachment by BVDV. In a recent study, Day-7 embryos were collected from superovulated cows, hatched in vitro, exposed to cytopathic or noncytopathic strains of BVDV, and subsequently cultured for up to 14 d (8). Cytopathic effect was observed in those that were exposed to cytopathic virus, but those that were exposed to noncytopathic BVDV expanded normally when compared to nonexposed controls. Infection with noncytopathic BVDV was confirmed in the apparently normal, developing embryos at the end of in vitro culture (8). Thus, zona pellucida-free in vivo-derived bovine
Theriogenology
embryos were susceptible to infection with BVDV, and infection with the noncytopathic virus did not have an apparent effect on development. Systems for in vitro production of bovine embryos do provide the opportunity for close and continuous observation of oocytes, the conceptus, and support cells; therefore, infectious agents causing observable abnormalities in support cells could be detected, and agents providing a total block to fertilization or development would not constitute an epidemiologic hazard since no embryos would be produced for transfer. However, infectious agents that do not cause observable cytopathic effects or do not dramatically reduce fertilization or embryonic development could easily escape detection and are of serious concern. In our earlier study, zona pellucida-free bovine embryos continued to develop without observable changes after infection with noncytopathic biotypes of BVDV (8). Preliminary data (unpublished) indicated that the SD-l isolate of noncytopathic BVDV would cause infections of bovine oviductal epithelial cells that were inapparent after short-term in vitro culture. Thus, the SD-l isolate was selected for use in this study. Isolation of the noncytopathic BVDV from all exposed oviductal epithelial cells confirmed that the virus was in the system during the time of fertilization and throughout the period of in vitro culture for treated groups of oocytes and presumptive zygotes/embryos, respectively. Failure to isolate the virus from all unexposed oviductal epithelial cells, provided reassurance that the virus had not been introduced inadvertently (via reagents or tissues) into the in vitro fertilization or in vitro culture drops of control oocytes and presumptive zygotes. All commercial lots of estrous cow serum that we had tested contained antibody to BVDV. Therefore, we elected to use estrous cow serum with anti-BVDV antibody in the in vitro culture medium, since this was representative of the type of serum likely to be used routinely in in vitro fertilization in our laboratory as well as in some other laboratories. Because specific antibody was present, it is not surprising that the virus never was isolated from medium collected from in vitro culture drops despite the presence of infected oviductal epithelial cells in all of these drops. However, it was initially surprising that we were only able to isolate the virus from the fertilization-TALP from 60% of the fertilization drops when infected oviductal epithelial cells were present in all of these drops. This finding is indicative of the presence of a component (or multiple components) in the in vitro fertilization medium with an inhibitory effect on the infectivity of the virus. Results of our additional preliminary study (not included) showed that the presence of bovine serum albumin was associated with reduced levels of free infectious BVDV in the fertilization-TALP. Although we have no proof, it is possible that immunoglobulin contamination of the bovine serum albumin was responsible for this anti-BVDV activity.
Theriogenology
851
Obviously, the anti-BVDV antibody in the in vitro culture medium and the undefined anti-BVDV activity in the fertilizationTALP, limited the exposure of oocytes and zygotes to free infectious virus during in vitro fertilization and in vitro However, these obstacles did not provide culture, respectively. total protection, since the virus was isolated from two groups of The mechanism by which the BVDV degenerated ova after washing. Virus was associated with these degenerated ova is uncertain. might have been carried across the zona pellucida at the time of fertilization or it could have become attached to or penetrated the zona pellucida at any time during the period of in vitro Regardless, it is important to note that these virusculture. positive ova were zona pellucida-intact and washed according to procedures that would have been effective for insuring that in vivo-derived zona pellucida-intact embryos were free of BVDV. Certainly, there is a need to wash in vitro-derived embryos: however, in light of our findings, it appears that additional precautions might have to be taken to insure freedom of these embryos from BVDV. The cleavage and development rates for both treated and control groups were slightly lower than expected from similar This may applications of these techniques in our laboratory. have been due to the additional time outside of the environment of the incubator that was required for separate handling of the treatment groups. Regardless, rates of cleavage and development were sufficient to allow statistical and visual comparisons that indicated no difference between groups of oocytes and presumptive zygotes fertilized and cultured in the presence or absence of oviductal epithelial cells that were infected with noncytopathic BVDV. It was not surprising to us that the presence of noncytopathic BVDV in oviductal epithelial cells was not visually apparent and did not result in differences in rates of cleavage and development; however, these results do raise some concern. As stated previously, key animal tissues (e.g., oocytes, cumulus cells, and oviductal epithelial cells) or sera used in the in vitro production of bovine embryos provide the potential for introduction of BVDV (7,13), yet guidelines for quality control of these components of in vitro fertilization systems have not been established. It was encouraging that no virus was isolated from morulae or blastocysts (that were of transferrable quality) after washing, and it is tempting to speculate that the isolations from degenerate ova may have been the result of "lethal infections IIthat do not constitute an epidemiological hazard since these embryos are not available for transfer. However, the number of viable embryos that were assayed was small, and‘it is possible that testing of additional morulae and blastocysts might have resulted in some positives. In this study, noncytopathic BVDV was introduced into a representative system for in vitro production of bovine embryos in a preliminary attempt to investigate epidemiological hazards
Theriogenology
852
associated with the virus. Infections of oviductal epithelial cells were not readily apparent and did not affect rates of cleavage and development or the apparent quality of morulae and blastocysts that were produced: therefore, there is a need for specific quality control measures to insure that systems for in vitro production of bovine embryos do not contain noncytopathic BVDV. Based on the current study, the.efficacy of using washing procedures recommended for insuring that in vivo-derived bovine embryos are free of BVDV to insure that in vitro-derived embryos are free of the virus has to be questioned. Additional work will be required to properly evaluate washing procedures for in vitroderived embryos.
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