212EFFECT of Cryopreservation Methods and Pre-Cryopreservation Storage on Bottlenose Dolphin (Tursiops Truncates) Spermatozoa (original) (raw)
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Theriogenology, 2007
The objectives of this study were to develop techniques to detect BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using either virus isolation (VI) or real time quantitative polymerase chain reaction (RT-QPCR) assays. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed to SD-1, a high affinity strain of BVDV, for 2 h and then processed according to the International Embryo Transfer Society (IETS) guidelines prior to testing. Groups of five or two in vivo-derived embryos, and single in vivo-derived embryos, were VI positive for BVDV 100, 50, and 33% of the time, and were RT-QPCR positive 100, 75, and 42% of the time, respectively. The virus was detected by the VI technique in all of the groups of five or two in vitro-produced embryos and in all of the single in vitro-produced embryos, and it was detected in 100, 80, and 50%, using RT-QPCR. Techniques for RT-QPCR were sufficiently sensitive to detect 10 copies of viral RNA in a sample and to detect BVDVassociated with single embryos. Application of this new technology, RT-QPCR, will facilitate additional studies to further assess the risk of transmission of BVDV through embryo transfer.
Theriogenology, 2017
As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 Â 10 2-2.50 Â 10 3 TCID 50 /100 mL), BVDV-2 (6.25 Â 10 1-5.20 Â 10 2 TCID 50 /100 mL) or Hobi-like virus (1.90 Â 10 2-1.58 Â 10 3 TCID 50 /100 mL) for 22e24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50e100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2e64.1% vs 49.8%; P ¼ 0.003e0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20e50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3e38.0% vs 49.8% for negative controls; P 0.001e0.014). Blastocyst rates were only reduced in the 7.9 Â 10 2 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P ¼ 0.009), when calculated by oocyte number. 50e80% of degenerated embryos tested positive for Hobilike virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and-2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.
Reproduction, 1995
Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus\p=n-\oocytecomplexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation. Viral titres present in oviduct, ovary and blood were between \m=ge\102.2 and 107; \m=ge\102.2 and 106.75; and 103.5 and 104.25 tissue culture infective doses, (TCID)50 g \ m=-\ 1, respectively. Control tissues from cattle not infected with BVDV, tested in each of the preceding techniques, were negative. These data establish that ovary and oviduct of persistently infected animals harbour non-cytopathogenic BVDV and that granulosa cells and oviduct epithelial cells, which are used as co-culture cells during bovine embryonic development in vitro and which, in the case of granulosa cells, constitute the cumulus investment surrounding the oocyte, are a vehicle for the potential transmission of BVDV to developing embryos.
Brazilian Journal of Microbiology, 2020
Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5′UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.
Most isolates of BVDV cause unapparent infections in cultured cells. Fetuses, postnatal animals or fetal bovine serum are possible sources of the virus for cultivated cells used as karyoplasts in cloning. Routine screening by veterinary diagnostic laboratories of 39 fetal fibroblast cell lines used in cloning research had revealed that 15 (39%) were positive for BVDV by various assays including RT-nPCR. As some were valuable transgenic cell lines, a rigorous protocol for evaluation of each line was undertaken to confirm infection with BVDV. A cryopreserved vial of each line was thawed, medium discarded and cells incubated (38.5 • C in 5% CO 2 and air) through 2 passages (6-10 days) in α-MEM supplemented with 10% equine serum. At the end of the second passage, cells were separated from medium, washed and assayed for presence of BVDV using virus isolation in 2 sequential passages in Madin Darby Bovine Kidney Cells and RT-nPCR. Available lots of fetal bovine serum and medium that had been used to culture the cells also were tested for BVDV. When the virus was detected, the RT-nPCR products were sequenced and compared. Also, an attempt was made to evaluate the earliest available cryopreserved passage of any positive cell lines. Results indicated that just 5 of 39 of the original cells tested (13%) were positive. Since cryopreserved earlier passages of 4 of the cell lines were available, they were assayed with the result that 2 of the 4 were not infected at the earliest passage. Further, BVDV was isolated from one lot of fetal bovine serum that was used to culture one of the cell lines. Sequence analysis verified that only 2 of these 4 cell lines were infected with the same isolate of BVDV, and one isolate was identical to the virus found in the fetal bovine serum used in medium to culture it. The discrepancy between our viral detection and that of the diagnostic laboratories is explained in part by the presumed test protocols. All BVDV-positive cells, as reported by the diagnostic laboratories, were positive by RT-nPCR. We presume that they did not separate medium from cells before assays. Thus, any noninfectious viral RNA that was in the medium (e.g. as would be expected in many lots of irradiated serum) would have been reported positive. The only possible sources for BVDV in these cell lines were the fetuses from which they originated or fetal bovine serum used in medium. Sequence analysis confirmed that serum was the source of viral infection in one line. The likely source of virus for 2 other lines was serum, since they were not infected at earlier passages. The 2 remaining cell lines were positive at the earliest available passages, so the fetuses from which cells were harvested could not be discounted as the source of BVDV. This report highlights the risks of introducing BVDV in embryo technologies and the difficulties that can be encountered in attempting accurate diagnosis of the presence of infectious virus.