- Email: [email protected]
Experimental collection and transfer of embryos from bovine immunodeficiency virus (BIV) infected cattle
ELSEVIER
EXPERIMENTAL COLLECTION AND TRANSFER OF EMBRYOS FROM BOVINE IMMUNODEFICIENCY VIRUS (BIV) INFECTED CATTLE A. Bielanski, la S. Nadin-Davis, 1 C. Simard, 3 p. Maxwell2 and J. Algire t IAnimal Disease Research Institute, Germplasm Centre of Expertise, Nepean, Ontario 2Centre for Animal and Plant Health, Retrovirology Centre ofExpertise,Charlottetown, PEI 3Health of Animals and Food Laboratory, St. Hyacinthe, Quebec, Canada Received for publication: December 9, 1999 Accepted: June 21, 2000 ABSTRACT Three experiments were conducted to determine whether the lentivirus, bovine immunodeficiency virus (BIV) is likely to be transmitted via embryo transfer. In the first experiment, embryos collected from BIV-negative heifers were exposed in vitro to BIV for 24 h, washed and then tested for the presence of the provirus. In the second experiment, embryos obtained from BIV-negative heifers were transferred to the uterine horns of BIVinfected heifers; 24 h later these embryos were recovered and tested for the presence of BIV. In the third experiment, embryos were collected from heifers experimentally infected with BIV and then transferred to BIV-negative recipients. In all three experiments, (BIV) proviral DNA was not detected by PCR in association with any oocytes, embryos, follicular fluid, oviductal or uterine washes. Twelve single embryos collected from BIV experimentally infected donors were transferred to BIV-negative recipients resulting in the birth of 7 calves all of which were also negative for BIV; the recipients remained BIV-negative throughout the experiment. In conclusion, this study demonstrates that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are unlikely to transmit this agent either to the recipients or the resulting offspring. © 2001 by Elsevier Science Inc.
Key words: bovine immunodeficiency virus, embryo transfer, embryos, in vitro fertilization
Acknowledgments The authors thank G.Raby and S. Chang for their technical support. a Correspondence and reprint request: Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario, Canada K2H 8P9 e-mail:[email protected] Theriogenology 55:641-648, 2001 © 2001 Elsevier Science Inc.
O093-691X/O1/$-see front matter PII: S0093-691X(01)00432-0
Theriogenology
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INTRODUCTION Bovine immunodeficiency virus (BIV), an RNA virus first isolated in the USA in 1969 from a cow R29 (18) with lymphocytosis and progressive emaciation (5), is a member of the lentivirus family and is thus closely related to the human immunodeficiency virus (HIV). These viruses cause persistent and usually lifelong infections in their host as a consequence of the integration of a proviral DNA into the genome of susceptible cells. Although prevalent worldwide, BIV has yet to be clearly associated with overt health problems in cattle (1, 14). However BIV may have a negative economic impact on the dairy industry (8). Recently, BIV provirus was detected by PCR in the white blood cells of 82% of samples of cryopreserved semen randomly selected from stud bulls in the USA (11) causing concern to both the AI and embryo transfer industries. Since it appears that BIV replicates in most if not all leucocyte sub-populations (7, 19) and that uterine flushes resulting from embryo collection may often contain blood cells, this study was performed to determine 1) whether replicating virus is present in the cells of the reproductive tract and embryos of infected embryo donors and 2) whether BIV can be transmitted by embryos collected from infected donors to BIV-negative recipients. MATERIAL AND METHODS Animals Fifteen holstein heifers 1.5 to 2 years of age were inoculated intravenously with 5 mL of BIV R29 isolate culture suspension (105 TCIDs0/mL). Whole blood samples were collected weekly for the first month, then at 2 wk intervals for the next 6 wk when the embryos were collected and at slaughter to confirm BIV infection. Genomic DNA was extracted from white blood cells and tested for the presence ofBIV proviruses by nested-PCR (10). Blood samples were collected at the intervals described above from all animals regardless of whether embryos were collected from them. Some of these animals were used as embryo donors ( Experiment 3) or as embryo recipients (Experiment 2). Another five heifers, inoculated with 5 mL of tissue culture medium from non-infected FBL cells, were used as a negative control group. These animals were tested for proviral DNA in the same manner as the infected group of heifers. They were used as the "clean" embryo donors (Experiment 1) and later postmortem for of "clean" IVF embryos (Experiment 2). A separate group of uninfected heifers (n = 12) served as embryo recipients (Experiment 3). Experiment 1. Exposure of Embryos to BIV In Vitro Five (BIV-negative) heifers were superovulated with a total dose of 400 mg of FSH-P (Folltropin, Vetrepharm, Ontario) over 4 d using a decreasing dose regime. On Day 7 after insemination, embryos were collected nonsurgically by washing the uterine horns each with 250 mL of phosphate buffered saline (PBS) supplemented with 2% estrous cow serum (ECS)
Theriogenology
643
and antibiotics. Serum used for the studies was collected from the BIV-negative group of animals which were found to be negative by nested PCR and free of BIV antibody on gagindirect ELISA (unpublished data). The embryos in the wash fluids were collected by filtration through a 75 ~tm filter membrane in an Emcon (Immuno System Inc., Spring Valley, WI, USA) system. The embryos thus collected on the membrane were retrieved by washing the filter membrane with 25 mL PBS supplemented with 2% ECS. Collected embryos then were exposed to BIV 105 TCIDs0/mL (Tissue Culture Infectious Doses/mL) culture in TCM-199 medium supplemented with 10% serum under 5% CO2 at 38°C for 24 h. The developmental status of the embryos was then examined microscopically before washing 10 times in PBS and assay for proviral BIV. Experiment 2. Exposure of Embryos to BIV In Vivo IVF embryos were produced from oocytes collected from uninfected heifers. Briefly, cumulus-oocyte complexes (COC) were aspirated from follicular fluid, matured in TCM-199 for 24 h and then were inseminated with motile frozen-thawed sperm fraction (tested free of proviral DNA) obtained by "swim up" procedure (13). Presumptive zygotes were cultured for 7 d on monolyars of cumulus cells in TCM-199. ZP-intact embryos at the morula and blastocyst stage in groups of 10 to 15 were transferred to the uterine horns of 5 experimentally infected BIV heifers on Day 7 of the estrus cycle. After 24 h embryos were recovered nonsurgically from the reproductive tract, washed 10 times in PBS and tested for proviral DNA. Experiment 3. Embryo Transfer Only heifers in which proviral DNA was detected were superovulated and embryos were collected as described in experiment 1. The first embryo collections began at 4 wk post-viral inoculation of heifers. The nonsurgical embryo collection was repeated 2 to 4 times on some heifers over a period of 47 wk post-viral inoculation (p.i.). After washing, good quality single embryos were selected and transferred nonsurgically into synchronized BIV-negative recipients on Day 7 or 8 of the estrus cycle. Pregnant recipients were housed in an isolation unit until calving at which time both mother and calf were tested for proviral DNA. At the end of the experiments, all BIV-infected heifers were slaughtered and follicular fluid, uterine washes, oviductal washes and oocytes were collected and tested for proviral DNA by PCR. No viral isolation or RNA extraction on those samples was attempted.
Embryo Washing The washing procedure consisted of transferring the oocytes and embryos by a semi automatic pipette with a 5~L disposable plastic tip to a petri dish containing 2 mL (dilution
Theriogenology
644
1:400) of fresh medium (PBS supplemented with 100 #g/mL ofpolyvinyl alcohol (Sigma, P8136), then gently swirling the dish with embryos for a few seconds. A new pipette tip was used for each transfer. Before the virus assay, groups of 10 or less oocytes/embryos were washed sequentially 10x and then subjected to the washing regimen normally used for the production of IVF embryos as described above. BIV Stock Preparation The BIV (isolate R29) was grown in fetal bovine lung (FBL) cells in TCM-199 supplemented with 10% FCS. The supematant was collected when cells were showing evident cytopathic effect, manifested by giant syncytium formation and cell senescence. The collected supematant was clarified by centrifugation at 5000 rpm for 10 min, then was filtered through a 22 ~m filter and frozen at -80°C before the inoculation of cattle. The virus stock was titrated by end-point dilution on FBL cells. The TCID 50% was calculated for the viral stock. Supematant from noninfected FBL cells was processed at the same time and was used for inoculation of the noninfected cattle group. BIV Proviral Assay Peripheral blood mononuclear cells (PBMCs) were recovered from whole blood after red blood cell lysis and cellular DNA was prepared by standard methods using phenol:chloroform extraction followed by ethanol precipitation (15). Similarly, DNA was purified from follicular fluid and IVF embryos collected from both BIV inoculated and noninfected control animals after slaughter. For the commercially slaughtered cattle, a pool of foUicular fluid and IVF embryos was employed for DNA extraction. Nested PCR, targeting portions of the BIV gag or pol genes, was performed on the DNA samples according to NadinDavis (10). The suitability of all DNA samples for amplification was verified using a portion of the [3-globin gene (9). Positive and negative controls consisted of DNA extracted from BIV (R29)-infected and noninfected FBL cell cultures respectively. Also, to ensure that there was no cross-contamination between the samples, vials without DNA were processed at the same time. RESULTS Inoculation of all cattle with BIV led to a persistent infection as determined by PCR analysis of PBMC DNA of these animals. While BIV infection was confirmed in most animals (10) by Week 3 post inoculation, detection was delayed in the others and one animal did not yield a positive result until Week 9 post inoculation. The infections were persistent until the slaughter of animals. The BIV proviral DNA was not detected in any of the blood samples taken from control uninfected animals. None of the infected or control animals showed any clinical signs of disease during the course of the experiments.
Theriogenology
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Experiment 1 A total of 44 morulae and blastocysts was collected from uninfected heifers and exposed in vitro to BIV for 24 h. After embryo washing 9 samples, each containing 3 to 5 ZPintact embryos, all tested negative for proviral DNA Experiment 2 Ten to 15 morula and blastocyst stage embryos fertilized in vitro were transferred to the uterus of each of 5 BIV infected heifers. Of a total of 62 transferred embryos, 43 were nonsurgically recovered. All samples containing embryos and uterine washes fromindividual heifers tested negative for proviral DNA. Experiment 3 A total of 147 embryos were recovered from 8 of the 15 infected animals by nonsurgical flushing. This procedure was repeated successfully 2 to 4 times on 6 of these animals over a period of 4 to 47 wk post-viral inoculation and subsequent proviral DNA detection. Embryonic development ranged from unfertilized eggs to expanded blastocyst and was similar to that observed for embryos collected from a control group of uninfected heifers. Seven heifers did not yield embryos; either they did not respond to the superovulatory treatment or embryos were not successfully recovered from them for unknown reason. All samples (n=16) containing embryos and corresponding uterine washes (n=12) tested negative for proviral DNA. In addition, at the end of the experiment, follicular fluid, oviductal fluid and samples containing oocytes collected post-mortem from these heifers were negative for proviral DNA. During the course of this study, 12 embryos collected from infected heifers were selected for transfer to BIV-negative recipients. Each recipient heifer received a single morula or blastocyst. Subsequently, seven heifers were deemed pregnant as determined by palpation or ultrasonography on Day 35 post-transfer, all bore developmentally normal but overweight calves at term. Blood from recipients and calves tested negative for proviral DNA. The remaining five nonpregnant heifers remained negative for BIV after unsuccessful embryo transfer. DISCUSSION In this study the standard methodology was used for embryo production and experimental design as recommended by lETS. Both in vitro and in vivo exposure of embryos to an infectious agent were used to determine their association with that infectious agent (3). During these experiments it was observed that BIV has no apparent effect on early embryonic development and that the infectious virus was not associated with ZP-intact
646
Theriogenology
embryos which were washed as recommended by the Intemational Embryo Transfer Society (17). Moreover, proviral DNA was not detected in the samples recovered from reproductive fluids indicating that the infectious virus was not infecting and integrating in the lymphocytes, granulosa cells, oviductal or uterine epithelial cells. From a practical standpoint this suggests that not only embryos but also oocytes from infected cows may be recovered for IVF free of integrated BIV. This view was reinforced by Experiment 2 in which clean IVF embryos were transferred for a short time to the reproductive tract of infected recipients and tested negative for proviral DNA after their retrieval. Although it remains beyond the scope of the present studies, it is interesting to note that natural transplacental infection of BIV was reported recently (16). Approximately 40% percent of seropositive cows gave birth to seropositive calves. Also, since the samples collected in the present study were not tested for viral RNA, it is not known whether free virus was associated with them or was present in the reproductive tract. Nevertheless, embryo transfer experiments proved that an infectious form of BIV was not transmitted to the recipients or their offspring. However, other retroviruses have been detected in uterine washes collected from seropositive cattle. For example, bovine leukemia virus (BLV) which was not detected directly from uterine tubal (UT) cells recovered from uterine washes was found in accompanying uterine lymphocytes, in contrast to bovine syncytial virus (BSV) which was detected in association with both UT cells and lymphocytes collected from seropositive animals (4). In this regard, it is possible that UT cells are not permissive to BIV in vivo as also reported for BLV (4). The lack of apparent effect of BIV on the health status of embryos mirrors observations made with other retroviruses. For example, it has been reported that embryos exposed in vitro or collected from animals seropositive to BLV (6), maedi-visna virus (20), caprine arthritis-encephalitis virus (21), and sheep pulmonary adenomatosis (12) exhibited normal development and were rendered from the infectious virus by multiple washing. These agents also were not transmitted to recipients by ET. However, it is not known whether in vitro derived embryos will interact with retroviruses in the same manner as in vivo produced embryos. It has already been documented that some pathogenic agents adhere more easily to the ZP of IVF embryos and they cannot be removed from the embryos by a simple washing procedure (2). In conclusion, the results of the present study demonstrate that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are not likely to transmit the virus to recipients and resulting offspring.
Theriogenology
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REFERENCES 1. 2.
3.
4.
5. 6.
7. 8.
9.
10.
11. 12.
13.
14. 15.
Belloc C, Polack B, Schwartz-Comil I, Brownlie J, Levy D. Bovine immunodeficiency virus: facts and questions. Vet Res 1996; 27: 395-402. Bielanski A. Review on disease transmission studies in relationship to production of embryos by in vitro fertilization and to related new reproductive technologies. Biotech Advances 1997; 15: 633-656. Bielanski A, Hare WCD. Procedures for design and analysis of research on transmission of infectious disease by embryo transfer. In: Manual of the International Embryo Transfer Society, Savoy, IL, IETS, 1998; 143-151. Bouillant AMP, Ruckerbauer DM, Eaglesome MD, Samach BS, Singh EL, Hare WCD, Randall GBC. Attempt to isolate bovine leukemia and bovine syncytial viruses from blood, uterine flush fluid, unfertilized ova and embryos from infected donor cattle. Ann Rech Vet 1981; 12: 385-395. Gonda MA, Luther DG, Fong SE, Tobin GJ. Bovine immunodeficiency virus: molecular biology and virus-host interactions. Virus Res 1994; 32:155-181. Hare WCD, Mitchell D, Singh EL, Bouillant AMP, Eaglesome MD, Ruckerbauer EM, Bielanski A, Randall GB. Embryo transfer in relation to bovine leukemia virus control and eradication. Can Vet J 1985; 26: 231-234. Heaton PR, Johnstone P, Brownlie J. Investigation of the cellular tropism of bovine immunodeficiency-like virus. Res Vet Sci 1998; 65: 33-40. McNab WB, Jacobs RM, Smith HE. A serological survey for bovine immunodeficiency-like virus in Ontario dairy cattle and association between test results production records and management practices. Can J Vet Res 1994; 58:36-41. Nadin-Davis SA. Detection of bovine immunodeficiency-like virus by the polymerase chain reaction. In: Y Becker and G Darai (Eds) PCR Protocols for Diagnosis of Human and Animal Virus Diseases. 1995; 491-506. Nadin-Davis SA, Chang SC, Smith HE, Jacobs RM. Detection of bovine immunodeficiency-like virus by the polymerase chain reaction. J Virol 1993; 66: 4518-4524. Nash JW, Hanson LA, St Cyr-Coats K. Bovine immunodeficiency virus in stud bull semen. Am J Vet Res 1995; 56: 760-763. Parker BNJ, Wrathall AE, Saunders RW, Dawson M, Done SH, Francis PG, Dexter I, Bradley R. Use of embryo transfer to prevent transmission of sheep pulmonary adenomatosis. Theriogenology 1997; 47: 379. Parrish JJ, Susko-Parrish J, Leibfried-Rutledge ML, Critzer ES, Eystone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1996; 25: 591-600. St Cyr-Coats K, Pruett SB, Nash JW, Cooper C. Bovine immunodeficiencyvirus: incidence of infection in Mississippi dairy cattle. Vet Microbiol 1994; 42: 181-189. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory manual, 2noed., Cold Spring Harbor, NY. 1989.
648
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17. 18. 19.
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Theriogenology Scholl DT, Truax RE, Babtista JM, Ingawa K, Orr KA, O'Reilly KL, Jenny BF. Natural transplancental infection of dairy calves with bovine immunodeficiency virus and estimation of effect on neonatal health. Prey Vet Med 2000; 43: 239-252. Stringfellow D, Seidel SM. Manual of the International Embryo Transfer Society. Savoy, Ill. 1998. Van der Maaten MJ, Boothe AD, Seger CL. Isolation of a virus from cattle with persistent lymphocytosis. J Natl Cancer Inst 1972; 49: 1649-1657. Whetstone CA, Suarez DL, Miller JM, Pesch BA, Harp JA. Bovine lentivirus induces early transient b-cell proliferation in experimentally inoculated cattle and appears to be pantropic. J Virol 1977; 71: 640-644. Woodall CJ, Mylne J, McKelvey WAC, Watt NJ. Polymerase chain reaction (PCR) as a novel method for investigating the transmission of Maedi-Visna virus (MVV) by preimplantation embryos. Proc. 3 rd Int Sheep Vet Conf, Edinburgh, Scotland, June 1993. Wolfe DF, Nusbaum KE, Lauerman LH, Mysinger PW, Riddel MG, Putman MR, Shumway LS, Powe TA. Embryo transfer from goats seropositive for caprine arthritisencephalitis virus. Theriogenology 1987; 28:307-316.
EXPERIMENTAL COLLECTION AND TRANSFER OF EMBRYOS FROM BOVINE IMMUNODEFICIENCY VIRUS (BIV) INFECTED CATTLE A. Bielanski, la S. Nadin-Davis, 1 C. Simard, 3 p. Maxwell2 and J. Algire t IAnimal Disease Research Institute, Germplasm Centre of Expertise, Nepean, Ontario 2Centre for Animal and Plant Health, Retrovirology Centre ofExpertise,Charlottetown, PEI 3Health of Animals and Food Laboratory, St. Hyacinthe, Quebec, Canada Received for publication: December 9, 1999 Accepted: June 21, 2000 ABSTRACT Three experiments were conducted to determine whether the lentivirus, bovine immunodeficiency virus (BIV) is likely to be transmitted via embryo transfer. In the first experiment, embryos collected from BIV-negative heifers were exposed in vitro to BIV for 24 h, washed and then tested for the presence of the provirus. In the second experiment, embryos obtained from BIV-negative heifers were transferred to the uterine horns of BIVinfected heifers; 24 h later these embryos were recovered and tested for the presence of BIV. In the third experiment, embryos were collected from heifers experimentally infected with BIV and then transferred to BIV-negative recipients. In all three experiments, (BIV) proviral DNA was not detected by PCR in association with any oocytes, embryos, follicular fluid, oviductal or uterine washes. Twelve single embryos collected from BIV experimentally infected donors were transferred to BIV-negative recipients resulting in the birth of 7 calves all of which were also negative for BIV; the recipients remained BIV-negative throughout the experiment. In conclusion, this study demonstrates that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are unlikely to transmit this agent either to the recipients or the resulting offspring. © 2001 by Elsevier Science Inc.
Key words: bovine immunodeficiency virus, embryo transfer, embryos, in vitro fertilization
Acknowledgments The authors thank G.Raby and S. Chang for their technical support. a Correspondence and reprint request: Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario, Canada K2H 8P9 e-mail:[email protected] Theriogenology 55:641-648, 2001 © 2001 Elsevier Science Inc.
O093-691X/O1/$-see front matter PII: S0093-691X(01)00432-0
Theriogenology
642
INTRODUCTION Bovine immunodeficiency virus (BIV), an RNA virus first isolated in the USA in 1969 from a cow R29 (18) with lymphocytosis and progressive emaciation (5), is a member of the lentivirus family and is thus closely related to the human immunodeficiency virus (HIV). These viruses cause persistent and usually lifelong infections in their host as a consequence of the integration of a proviral DNA into the genome of susceptible cells. Although prevalent worldwide, BIV has yet to be clearly associated with overt health problems in cattle (1, 14). However BIV may have a negative economic impact on the dairy industry (8). Recently, BIV provirus was detected by PCR in the white blood cells of 82% of samples of cryopreserved semen randomly selected from stud bulls in the USA (11) causing concern to both the AI and embryo transfer industries. Since it appears that BIV replicates in most if not all leucocyte sub-populations (7, 19) and that uterine flushes resulting from embryo collection may often contain blood cells, this study was performed to determine 1) whether replicating virus is present in the cells of the reproductive tract and embryos of infected embryo donors and 2) whether BIV can be transmitted by embryos collected from infected donors to BIV-negative recipients. MATERIAL AND METHODS Animals Fifteen holstein heifers 1.5 to 2 years of age were inoculated intravenously with 5 mL of BIV R29 isolate culture suspension (105 TCIDs0/mL). Whole blood samples were collected weekly for the first month, then at 2 wk intervals for the next 6 wk when the embryos were collected and at slaughter to confirm BIV infection. Genomic DNA was extracted from white blood cells and tested for the presence ofBIV proviruses by nested-PCR (10). Blood samples were collected at the intervals described above from all animals regardless of whether embryos were collected from them. Some of these animals were used as embryo donors ( Experiment 3) or as embryo recipients (Experiment 2). Another five heifers, inoculated with 5 mL of tissue culture medium from non-infected FBL cells, were used as a negative control group. These animals were tested for proviral DNA in the same manner as the infected group of heifers. They were used as the "clean" embryo donors (Experiment 1) and later postmortem for of "clean" IVF embryos (Experiment 2). A separate group of uninfected heifers (n = 12) served as embryo recipients (Experiment 3). Experiment 1. Exposure of Embryos to BIV In Vitro Five (BIV-negative) heifers were superovulated with a total dose of 400 mg of FSH-P (Folltropin, Vetrepharm, Ontario) over 4 d using a decreasing dose regime. On Day 7 after insemination, embryos were collected nonsurgically by washing the uterine horns each with 250 mL of phosphate buffered saline (PBS) supplemented with 2% estrous cow serum (ECS)
Theriogenology
643
and antibiotics. Serum used for the studies was collected from the BIV-negative group of animals which were found to be negative by nested PCR and free of BIV antibody on gagindirect ELISA (unpublished data). The embryos in the wash fluids were collected by filtration through a 75 ~tm filter membrane in an Emcon (Immuno System Inc., Spring Valley, WI, USA) system. The embryos thus collected on the membrane were retrieved by washing the filter membrane with 25 mL PBS supplemented with 2% ECS. Collected embryos then were exposed to BIV 105 TCIDs0/mL (Tissue Culture Infectious Doses/mL) culture in TCM-199 medium supplemented with 10% serum under 5% CO2 at 38°C for 24 h. The developmental status of the embryos was then examined microscopically before washing 10 times in PBS and assay for proviral BIV. Experiment 2. Exposure of Embryos to BIV In Vivo IVF embryos were produced from oocytes collected from uninfected heifers. Briefly, cumulus-oocyte complexes (COC) were aspirated from follicular fluid, matured in TCM-199 for 24 h and then were inseminated with motile frozen-thawed sperm fraction (tested free of proviral DNA) obtained by "swim up" procedure (13). Presumptive zygotes were cultured for 7 d on monolyars of cumulus cells in TCM-199. ZP-intact embryos at the morula and blastocyst stage in groups of 10 to 15 were transferred to the uterine horns of 5 experimentally infected BIV heifers on Day 7 of the estrus cycle. After 24 h embryos were recovered nonsurgically from the reproductive tract, washed 10 times in PBS and tested for proviral DNA. Experiment 3. Embryo Transfer Only heifers in which proviral DNA was detected were superovulated and embryos were collected as described in experiment 1. The first embryo collections began at 4 wk post-viral inoculation of heifers. The nonsurgical embryo collection was repeated 2 to 4 times on some heifers over a period of 47 wk post-viral inoculation (p.i.). After washing, good quality single embryos were selected and transferred nonsurgically into synchronized BIV-negative recipients on Day 7 or 8 of the estrus cycle. Pregnant recipients were housed in an isolation unit until calving at which time both mother and calf were tested for proviral DNA. At the end of the experiments, all BIV-infected heifers were slaughtered and follicular fluid, uterine washes, oviductal washes and oocytes were collected and tested for proviral DNA by PCR. No viral isolation or RNA extraction on those samples was attempted.
Embryo Washing The washing procedure consisted of transferring the oocytes and embryos by a semi automatic pipette with a 5~L disposable plastic tip to a petri dish containing 2 mL (dilution
Theriogenology
644
1:400) of fresh medium (PBS supplemented with 100 #g/mL ofpolyvinyl alcohol (Sigma, P8136), then gently swirling the dish with embryos for a few seconds. A new pipette tip was used for each transfer. Before the virus assay, groups of 10 or less oocytes/embryos were washed sequentially 10x and then subjected to the washing regimen normally used for the production of IVF embryos as described above. BIV Stock Preparation The BIV (isolate R29) was grown in fetal bovine lung (FBL) cells in TCM-199 supplemented with 10% FCS. The supematant was collected when cells were showing evident cytopathic effect, manifested by giant syncytium formation and cell senescence. The collected supematant was clarified by centrifugation at 5000 rpm for 10 min, then was filtered through a 22 ~m filter and frozen at -80°C before the inoculation of cattle. The virus stock was titrated by end-point dilution on FBL cells. The TCID 50% was calculated for the viral stock. Supematant from noninfected FBL cells was processed at the same time and was used for inoculation of the noninfected cattle group. BIV Proviral Assay Peripheral blood mononuclear cells (PBMCs) were recovered from whole blood after red blood cell lysis and cellular DNA was prepared by standard methods using phenol:chloroform extraction followed by ethanol precipitation (15). Similarly, DNA was purified from follicular fluid and IVF embryos collected from both BIV inoculated and noninfected control animals after slaughter. For the commercially slaughtered cattle, a pool of foUicular fluid and IVF embryos was employed for DNA extraction. Nested PCR, targeting portions of the BIV gag or pol genes, was performed on the DNA samples according to NadinDavis (10). The suitability of all DNA samples for amplification was verified using a portion of the [3-globin gene (9). Positive and negative controls consisted of DNA extracted from BIV (R29)-infected and noninfected FBL cell cultures respectively. Also, to ensure that there was no cross-contamination between the samples, vials without DNA were processed at the same time. RESULTS Inoculation of all cattle with BIV led to a persistent infection as determined by PCR analysis of PBMC DNA of these animals. While BIV infection was confirmed in most animals (10) by Week 3 post inoculation, detection was delayed in the others and one animal did not yield a positive result until Week 9 post inoculation. The infections were persistent until the slaughter of animals. The BIV proviral DNA was not detected in any of the blood samples taken from control uninfected animals. None of the infected or control animals showed any clinical signs of disease during the course of the experiments.
Theriogenology
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Experiment 1 A total of 44 morulae and blastocysts was collected from uninfected heifers and exposed in vitro to BIV for 24 h. After embryo washing 9 samples, each containing 3 to 5 ZPintact embryos, all tested negative for proviral DNA Experiment 2 Ten to 15 morula and blastocyst stage embryos fertilized in vitro were transferred to the uterus of each of 5 BIV infected heifers. Of a total of 62 transferred embryos, 43 were nonsurgically recovered. All samples containing embryos and uterine washes fromindividual heifers tested negative for proviral DNA. Experiment 3 A total of 147 embryos were recovered from 8 of the 15 infected animals by nonsurgical flushing. This procedure was repeated successfully 2 to 4 times on 6 of these animals over a period of 4 to 47 wk post-viral inoculation and subsequent proviral DNA detection. Embryonic development ranged from unfertilized eggs to expanded blastocyst and was similar to that observed for embryos collected from a control group of uninfected heifers. Seven heifers did not yield embryos; either they did not respond to the superovulatory treatment or embryos were not successfully recovered from them for unknown reason. All samples (n=16) containing embryos and corresponding uterine washes (n=12) tested negative for proviral DNA. In addition, at the end of the experiment, follicular fluid, oviductal fluid and samples containing oocytes collected post-mortem from these heifers were negative for proviral DNA. During the course of this study, 12 embryos collected from infected heifers were selected for transfer to BIV-negative recipients. Each recipient heifer received a single morula or blastocyst. Subsequently, seven heifers were deemed pregnant as determined by palpation or ultrasonography on Day 35 post-transfer, all bore developmentally normal but overweight calves at term. Blood from recipients and calves tested negative for proviral DNA. The remaining five nonpregnant heifers remained negative for BIV after unsuccessful embryo transfer. DISCUSSION In this study the standard methodology was used for embryo production and experimental design as recommended by lETS. Both in vitro and in vivo exposure of embryos to an infectious agent were used to determine their association with that infectious agent (3). During these experiments it was observed that BIV has no apparent effect on early embryonic development and that the infectious virus was not associated with ZP-intact
646
Theriogenology
embryos which were washed as recommended by the Intemational Embryo Transfer Society (17). Moreover, proviral DNA was not detected in the samples recovered from reproductive fluids indicating that the infectious virus was not infecting and integrating in the lymphocytes, granulosa cells, oviductal or uterine epithelial cells. From a practical standpoint this suggests that not only embryos but also oocytes from infected cows may be recovered for IVF free of integrated BIV. This view was reinforced by Experiment 2 in which clean IVF embryos were transferred for a short time to the reproductive tract of infected recipients and tested negative for proviral DNA after their retrieval. Although it remains beyond the scope of the present studies, it is interesting to note that natural transplacental infection of BIV was reported recently (16). Approximately 40% percent of seropositive cows gave birth to seropositive calves. Also, since the samples collected in the present study were not tested for viral RNA, it is not known whether free virus was associated with them or was present in the reproductive tract. Nevertheless, embryo transfer experiments proved that an infectious form of BIV was not transmitted to the recipients or their offspring. However, other retroviruses have been detected in uterine washes collected from seropositive cattle. For example, bovine leukemia virus (BLV) which was not detected directly from uterine tubal (UT) cells recovered from uterine washes was found in accompanying uterine lymphocytes, in contrast to bovine syncytial virus (BSV) which was detected in association with both UT cells and lymphocytes collected from seropositive animals (4). In this regard, it is possible that UT cells are not permissive to BIV in vivo as also reported for BLV (4). The lack of apparent effect of BIV on the health status of embryos mirrors observations made with other retroviruses. For example, it has been reported that embryos exposed in vitro or collected from animals seropositive to BLV (6), maedi-visna virus (20), caprine arthritis-encephalitis virus (21), and sheep pulmonary adenomatosis (12) exhibited normal development and were rendered from the infectious virus by multiple washing. These agents also were not transmitted to recipients by ET. However, it is not known whether in vitro derived embryos will interact with retroviruses in the same manner as in vivo produced embryos. It has already been documented that some pathogenic agents adhere more easily to the ZP of IVF embryos and they cannot be removed from the embryos by a simple washing procedure (2). In conclusion, the results of the present study demonstrate that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are not likely to transmit the virus to recipients and resulting offspring.
Theriogenology
647
REFERENCES 1. 2.
3.
4.
5. 6.
7. 8.
9.
10.
11. 12.
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