104 FAILURE TO REMOVE BLUETONGUE SEROTYPE 8 VIRUS (BTV-8) FROM IN VITRO-PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 166
Author(s):  
A. O. Penido ◽  
K. De Clerq ◽  
A. Haegeman ◽  
L. Vandaele ◽  
H. Nauwynck ◽  
...  
Keyword(s):  
Room Temperature ◽  
Virus Isolation ◽  
Emerging Diseases ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Trypsin Treatment ◽  
Virus Removal ◽  
Virus Serotype ◽  
Temperature Group

Bluetongue virus serotype 8 (BTV-8) causes some unique characteristics compared with other BTV strains, such as transplacentary transmission, infertility, and diminished health of the offspring (De Clercq et al. 2008 Transboundary and Emerging Diseases 55, 352–359), and concerns exist about the risk of the transmission of the disease via embryo transfer (Vandaele et al. 2012). It is known that most pathogenic agents can be eliminated by washing and trypsin treatment of intact embryos according to the IETS guidelines, but some viruses adhere strongly to the zona pellucida and are not removed by this process (Ali al Ahmad et al. 2011 Theriogenology 76, 126–132). The aim of this study was to investigate decontaminating methods for bovine in vitro embryos that had been infected in vitro with BTV-8, which were earlier shown to be effective in goat embryos (REF). In vitro bovine blastocysts (n = 105) were placed in 800 μL of minimal essential medium (MEM), containing 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO2 in air (Vandaele et al. 2011 Vet. Res. 42, 14–21). The embryos were exposed to trypsin either at 37°C [Group 1 (G1)] or at room temperature [Group 2 (G2)], with 3 treatments per group (5 embryos/treatment), consisting of 5 washes in PBS without BSA; 2 washes in 0.25% trypsin for 45 s each [treatment 1 (T1)], 2 washes in 0.25% trypsin-EDTA for 60 s each [treatment 2 (T2)], or 2 washes in 0.25% trypsin for 90 s each [treatment 3 (T3)]; and 10 washes in PBS + 0.4% BSA. All the treatments were done in triplicate. The efficiency of the different washing techniques and trypsin temperature for virus removal was evaluated by RT-quantitative PCR (qPCR) on embryos and washes. Virus isolation was performed on embryonated chicken eggs as described by Vandaele et al. (2011 Vet. Res. 42, 14–21) for the first and last washing fluids and for the embryos. Room temperature was 24.9°C. Viral BTV RNA was detected by RT-PCR in the first 5 washes in all groups and treatments. After the trypsin wash, all samples remained negative until the last wash procedure. Viral isolation was positive in the first 3 washes and negative in the 10th wash. The embryos were positive on RT-PCR in at least 2 replicates of each treatment, but all samples remained negative on virus isolation. The results show that the wash procedure is efficient to remove the virus from the wash media, but it failed to remove the virus from bovine embryos produced in vitro. The temperature (37°C or room temperature) did not influence the efficiency of the trypsin treatment.

174 THE EFFECT OF DIFFERENT OVARY TRANSPORT TEMPERATURES ON IN VITRO DEVELOPMENT AND POST-THAW SURVIVAL OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 203
Author(s):  
S. R. Cho ◽  
S. H. Choi ◽  
H. J. Kim ◽  
C. Y. Choe ◽  
H. J. Jin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Development Rate ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Embryo Freezing ◽  
Nuclear Maturation ◽  
Vitro Development ◽  
And Control

The present study was carried out to investigate the effect of different ovary transport temperatures on in vitro development and post-thaw survivability of bovine embryos. Bovine ovaries were collected at a local slaughterhouse and transported at 4 different temperature categories to the laboratory: 7–10�C (T1), 11–17�C (T2), 18–25�C (T3), and above 26�C (control group). The cumulus–oocyte complexes (COCs) were aspirated from 2–8 mm antral follicles using a syringe with an 18 gauge needle. Selected COCs were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 50 COCs were matured for 22 h in 4-well dishes of TCM-199 supplemented with 5% FBS, 10 �g mL-1 LH, and 10 �g mL-1 FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2 in air at 39�C. Mature COCs were fertilized with frozen–thawed semen treated with BO medium. To evaluate nuclear maturation to the metaphase II stage, the matured COCs were fixed in 1 : 3 acetic acid–ethanol for 30 s and stained with 3% basic Fuchsin. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.8 M ethylene glycol as a cryoprotectant. Embryos were loaded into 0.25-mL straws at room temperature, plunged directly into a cooling chamber, kept at -7�C for 10 min, including time for seeding, and further cooled to -35�C at -0.3�C min-1; after 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in a water bath at 37�C. The appearance of the embryos was evaluated immediately after warming and again at 24-h intervals for at least 3 days. The development rate was assessed by the re-expansion of the blastocoel and the hatching of blastocysts. Results were compared by ANOVA. The rates of maturation (to metaphase II), cleavage, and development to blastocysts were compared among treatment groups. Furthermore, frozen–thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2, and T3 groups (24/40, 60.0%; 25/41, 61.0%; and 30/44, 68.2%, respectively) were significantly lower than that in the control group (36/44, 81.8%; P < 0.05). The cleavage rates in the T1 and T2 groups (61/116, 52.6% and 66/121, 54.5%) were significantly lower than that in the control group (112/134, 83.6%; P < 0.05). However, there was no difference in the development rate to blastocysts among all groups (27.9–33.0%; P > 0.05). The survivability of frozen–thawed embryos was significantly lower in the T1 group (6/13, 46.2%) than in the T2 (11/16, 68.8), T3 (13/18, 72.2%), and control groups (19/26, 73.1%; P < 0.05). In conclusion, the results suggest that ovary transport at 26�C may be optimal for better in vitro development and survival of frozen–thawed embryos produced in vitro. Furthermore, exposure of ovaries to temperatures below 10�C during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.

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

Zygote ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 307-315 ◽  
Author(s):  
A.V. Makarevich ◽  
J. Pivko ◽  
E. Kubovicova ◽  
P. Chrenek ◽  
M. Slezaková ◽  
...  
Keyword(s):  
Embryo Development ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Trypsin Treatment ◽  
Cell Organelles ◽  
Virus Like Particles ◽  
Ultrastructural Studies ◽  
Morula Stage ◽  
Cellular Organelles

SummaryThe aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 106.16 TCID50/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR–FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles.It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.

Washing and trypsin treatment of in vitro derived bovine embryos exposed to bovine viral diarrhea virus

1998 ◽  
Vol 50 (5) ◽  
pp. 717-726 ◽  
Author(s):  
E Trachte ◽  
D Stringfellow ◽  
K Riddell ◽  
P Galik ◽  
M Riddell ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Bovine Embryos ◽  
Trypsin Treatment ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

143 PRESENCE OF PROSTAGLANDIN F2α RECEPTOR IN IN VITRO-DERIVED MORULA AND BLASTOCYST STAGE BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 180 ◽  
Author(s):  
F. N. Scenna ◽  
J. L. Edwards ◽  
G. M. Pighetti ◽  
F. N. Schrick
Keyword(s):  
Embryonic Development ◽  
Protein Expression ◽  
Room Temperature ◽  
Positive Control ◽  
Blastocyst Stage ◽  
Receptor Protein ◽  
Bovine Embryos ◽  
Pvdf Membrane ◽  
Receptor Mrna

Culture of in vitro and in vivo-derived embryos in medium containing prostaglandin F2� (PGF) decreased embryonic development to blastocyst stage and reduced hatching rates (Scenna et al. Prostaglandins 73, 215-226). Moreover, administration of an inhibitor of PGF synthesis at the time of embryo transfer in bovine recipients improved pregnancy rates (Schrick et al. 2001 Theriogenology 59, 335 abstr.). These findings indicate a direct negative effect of PGF on embryonic development. However, to our knowledge, no evidence of PGF receptor expression in morula or blastocyst stage bovine embryos is available in the literature. Therefore, the objective of the current study was to determine the presence of PGF receptor mRNA using real-time RT-PCR and protein expression by Western blotting in morula or blastocyst stage in vitro bovine embryos. Briefly, isolated total RNA from compact morula or blastocyst stage embryos and from bovine tongue epithelium (positive control for PGF receptor mRNA) were reverse-transcribed into cDNA. A volume from the RT reaction equivalent to 10 embryos per tube was utilized to determine transcripts for PGF receptor and Histone H2A (standard PCR control). Polymerase chain reaction was performed, and identity of PCR fragments was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis and by DNA sequencing. To determine protein expression, morula and blastocyst stage embryos were lysed in lysis buffer (10% SDS, 1 m Tris pH 7.5, 1 m NaF, 1 m DTT, 0.1 m EGTA with protease inhibitors) and stored at -20�C. Crude proteins isolated from bovine corpora lutea (positive control for PGF receptor protein), embryo samples, and prestained standards were separated by 12% SDS-PAGE under reducing conditions. Proteins were electrotransferred onto a PVDF membrane. Nonspecific binding sites in the PVDF membrane were blocked with 10% nonfat dry milk, and the blot was washed and incubated for 1 h at room temperature with a 1:1000 dilution of the primary antibody (rabbit polyclonal antibody against PGF receptor protein). Subsequently, the blot was washed and incubated for 1 h at room temperature with 1:1000 dilution of mouse anti-rabbit IgG conjugated with horseradish peroxidase. Finally, the blot was washed and revealed by chemiluminescence in a CCD camera. Results indicated that transcripts as well as the protein for PGF receptor were present in early stage bovine embryos. Identification of PGF receptor in morula and blastocyst stage bovine embryos may, in part, explain the increase in pregnancy rates after administration of a PGF synthesis inhibitor at the time of embryo transfer, which opens up the possibility to develop new strategies to prevent detrimental effects of PGF during early embryonic development.

192 SUSCEPTIBILITY OF BOVINE-HATCHED BLASTOCYSTS TO BLUETONGUE VIRUS SEROTYPE 8 INFECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 254
Author(s):  
L. Vandaele ◽  
W. Wesselingh ◽  
K. De Clercq ◽  
H. Nauwynck ◽  
A. Van Soom
Keyword(s):  
Virus Infection ◽  
Bluetongue Virus ◽  
Calf Serum ◽  
Virus Antigen ◽  
Emerging Diseases ◽  
Immunofluorescent Staining ◽  
Research Centre ◽  
Virus Serotype ◽  
Long Time

In 2006 and 2007, Bluetongue virus serotype 8 (BTV-8) caused devastating outbreaks in Northern Europe; the outbreaks were controlled in 2008 and 2009 by an international vaccination policy. Remarkably, BTV-8 differs from other serotypes in that it spread transplacentally (De Clercq K et al. 2008 Transboundary and Emerging Diseases 55, 352-359). Apart from the transplacental spreading, a significant increase in the incidence of abortions was reported in Belgium (Meroc E et al. 2009 Transboundary and Emerging Diseases 56, 39-48). The aim of the present study was to investigate the susceptibility of bovine-hatched, in vitro-produced blastocysts to BTV-8. A total of 1390 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes (n = 1148) were denuded 24 h post-insemination and cultured in 50-μL droplets of modified synthetic oviduct fluid (SOF) medium with 10% fetal calf serum (tested negative for BTV antibodies) at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. For virus incubation, BTV-8 Bel 2006/2 from Veterinary and Agrochemical Research Centre (VAR, Brussels, Belgium) was used. At 8.5 dpi, hatched embryos were placed in 800μL of minimum essential medium (MEM) containing 103.8 50% tissue culture infectious doses (TCID50) of BTV-8 and incubated for 1 h at 39°C in an atmosphere of 5% CO2 in air. At the same time, 2 groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After infection, all embryos were washed according to IETS guidelines with the exception that they were not zona pellucida intact and cultured in new SOF. At 48, 60, 72, and 96 h post-infection (hpi), one-fourth of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 with double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France). All control embryos (CTRL and MEM) were negative for BTV-8 virus antigen at all time points. At 48 hpi, only 1 out of 7 infected embryos was positive for virus antigen (in all its cells). At 60 hpi, all remaining embryos (n = 6) were negative, whereas at 72 hpi and 96 hpi all embryos had 25% to 100% BTV-8-positive cells (n = 6 at 72 hpi and n = 7 at 96 hpi). Furthermore, 1 embryo at 72 hpi and 2 embryos at 96 hpi showed morphological signs of degeneration. This study has showed for the first time that hatched in vitro-produced blastocysts are susceptible for BTV-8 virus infection and replication in vitro. The relatively long time between virus infection and detection of viral antigen is in accordance with the slow replication cycle of the virus. Further research is ongoing to investigate the importance of BTV-8 infection in early embryonic death. The first author is supported by Research Foundation-Flanders.

214 USE OF VIRUS ISOLATION AND QUANTITATIVE POLYMERASE CHAIN-REACTION TECHNIQUES TO DETECT BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN SINGLE OR SMALL GROUPS OF PRE-IMPLANTATION BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 214
Author(s):  
J. Waldrop ◽  
M. Givens ◽  
K. Riddell ◽  
P. Galik ◽  
D. Stringfellow
Keyword(s):  
Small Groups ◽  
Virus Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
Bovine Embryos ◽  
Chain Reaction ◽  
Diarrhea Virus ◽  
Polymerase Chain ◽  
Viral Diarrhea Virus

Because of its broad distribution among populations of cattle and its association with materials of animal origin used in embryo production, bovine viral diarrhea virus (BVDV) is a potential problem in applications of embryo technologies. While some isolates of BVDV are known to associate with both in vivo-derived and in vitro-produced bovine embryos, it has yet to be determined if the quantity of virus associated with exposed zona pellucida-intact embryos is sufficient to infect susceptible recipient cows via the intrauterine route. Techniques to detect and quantify BVDV associated with single transferable embryos are important to determine the risk of transmitting BVDV via embryo transfer. The objectives of this study were to define reproducible techniques to detect and quantify BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using virus isolation (VI) or real time quantitative polymerase chain reaction (Q-PCR) assays. In vivo-derived and in vitro-produced embryos were exposed for 2 h to approximately 106-cell culture infective doses (50% endpoint) per mililiter of a high affinity strain of BVDV, SD-1, and then washed according to IETS guidelines. Embryos were assayed in groups of five or two embryos, or single. There were 5 replicates of the group of five embryos, 4 of the group of two embryos, and 3 of the single embryos for the in vivo-derived embryos undergoing VI; 5, 4, and 2 replicates, respectively, undergoing Q-PCR, and 2, 5, and 2 replicates, respectively, for the in vitro-produced embryo groups undergoing VI and Q-PCR. Those to be assayed by VI were sonicated and the sonicate fluids were layered onto Madin Darby Bovine Kidney (MDBK) cells and passaged to allow for viral replication; an immunoperoxidase monolayer assay was then used for viral detection. A Roche� RNA/DNA extraction kit (Roche Diagnostic Systems, Inc., Somerville, NJ, USA) was used to extract RNA from virally exposed embryos, and extracted samples were assayed in duplicate Q-PCR reactions consisting of 100 �L. The primers used were L1 and U3 which are specific for conserved areas of the 5 prime nontranslated regions of the viral genome of BVDV. The PCR product was detected using hybridization probes s1 and s2 as in Struder et al. 2002 Biologicals 40, 289-296. In vivo-derived groups of five or two embryos, or single embryos, were positive for BVDV 100, 50, and 30% of the time, respectively, when VI was used and 100, 75 and 100%, respectively, when Q-PCR was used. The virus was detected in all of the in vitro-produced embryo groups of five, or two embryos, or single embryos, 100% of the time using VI, and in 100, 80, and 100% respectively, using Q-PCR. The virus isolation technique is highly sensitive but the need to destroy embryos by sonication to identify any embryo-associated virus precludes its use for embryos intended for transfer. Techniques for Q-PCR were sufficiently sensitive to detect and quantify 10 copies of RNA in a sample and to detect BVDV associated with single embryos.

112 BLUETONGUE VIRUS INFECTION IN CATTLE AFTER TRANSFER OF BOVINE IN VIVO-DERIVED EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 168 ◽  
Author(s):  
L. Vandaele ◽  
K. De Clercq ◽  
W. Van Campe ◽  
I. De Leeuw ◽  
A. Van Soom
Keyword(s):  
Embryo Transfer ◽  
Bluetongue Virus ◽  
Bovine Viral Diarrhoea Virus ◽  
Petri Dish ◽  
Bovine Embryos ◽  
Trypsin Treatment ◽  
Serum Samples ◽  
Diarrhoea Virus

Bluetongue virus (BTV) has been categorized by the OIE as a category 1 disease agent, for which proper handling between collection and transfer is thought to be sufficient to prevent transmission through embryo transfer. For bovine viral diarrhoea virus, it was shown that effectiveness of washing procedures depends on virus strains (Waldrop et al. 2004 Theriogenology 62, 45–55). Also BTV-8 has unique characteristics in comparison with other strains (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). The aim here was to investigate whether embryo transfer of in vivo-derived bovine embryos after in vitro exposure to BTV-8 can be performed without risk for infection of the recipients if IETS washing and trypsin treatment procedures are followed. Donor cows (n = 2) were synchronized and superovulated using Stimufol® (Ulg, Liége, Belgium) and subsequently inseminated. At 6.5 days post-insemination (dpi), flushed embryos (n = 14 and n = 3) were placed in 800 μL of minimal essential medium (MEM), containing 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO2 in air (Vandaele et al. 2011 Vet. Res. 42, 14–21). Next, embryos were washed in pairs in 5 consecutive Petri dishes containing PBS with antibiotics and 0.4% BSA, w/o Ca and Mg. Then, embryos were exposed to 2 consecutive trypsin (Invitrogen, Carlsbad, CA, 25050-014) washes of 45 s each at 39°C in 5% CO2 in air and finally, another 5 consecutive washes in PBS with 2% FCS. Each Petri dish contained at least 2 mL of medium and was gently agitated between washes. Embryos were transferred in a maximum of 7 μL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analysed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n = 6) were loaded in straws and transferred to 3 BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analysed for BTV-8 (RT-qPCR) and BTV-8 antibodies. Viral BTV-RNA was detected in all 3 recipient cows at 7 days after transfer and viraemia was confirmed by the establishment of high antibody titers at 14 days after transfer. Viral BTV-RNA was detected in washes 1 to 5 for each pair of embryos (Cp-value around 29), whereas washes 6 to 10 had Cp-values around the cut-off value (40), indicating that probably the last wash was BTV-8 negative. None of the recipients was pregnant at 28 days post-transfer. In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro-spiked in vivo-derived bovine embryos. These unexpected results stress the need for further in vivo research, e.g. what is the virus load in vivo embryos may be exposed to in utero during viraemia? Does BTV-8 react differently with the zona compared with other strains? Are alternative washing procedures needed to remove BTV-8 from the zona?

111 RISK OF CHLAMYDIA ABORTUS TRANSMISSION VIA EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 186
Author(s):  
F. Fieni ◽  
M. Oseikria ◽  
K. Laroucau ◽  
F. Vorimore ◽  
D. Tainturier ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Early Embryo ◽  
Control Group ◽  
Bovine Embryo ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Chlamydia Abortus ◽  
Zoonotic Risk

Chlamydia abortus (C. abortus) in cattle has been reported sporadically throughout the world and is implicated in respiratory, ocular, and reproductive disease as abortion, infertility, chronic mastitis, vaginal discharge, and endometritis. In addition, C. abortus presents a zoonotic risk exposure of pregnant women to infected animal and can lead to severe septicaemia in the mother, resulting in spontaneous abortion or stillbirth of the fetus. To investigate the risk of C. abortus transmission via bovine embryo transfer, our study aims to determine whether the embryonic ZP of in vitro-produced embryos protects early embryo cells against C. abortus infection and whether the bacteria adhere to or infect the cells of early bovine embryos (ZP-free) after in vitro infection. We also evaluated the efficacy of the washing procedure recommended by the IETS to decontaminate bovine embryos exposed to C. abortus in vitro. Ninety (8 to 16 cells) bovine embryos, produced in vitro, were randomly divided into 10 batches. Eight batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4.8 × 107 Chlamydia/mL of AB7 strain (ANSES, Maisons-Alfort, France). After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a PBS and 5% FCS solution without trypsin nor antibiotics in accordance with IETS guidelines. In parallel, 2 batches of 5 embryos (1 ZP-intact and 1 ZP-free) were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13 000 × g. The embryos and wash pellets were tested using RT-PCR. Chlamydia abortus DNA was found in all ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the tenth wash fluid for 1 batch (1/4) of ZP-intact infected embryos and in 3 batches (3/4) of ZP-free infected embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. These results demonstrate that C. abortus adheres to or penetrates the ZP as well as the early embryonic cells of in vitro-produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring. Nevertheless, the finding of C. abortus DNA by RT-PCR did not imply that the bacteria found is still infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. abortus would eliminate the bacteria from the ZP.

141 EXPRESSION STATUS OF AQUAPORINS 3, 7, AND 9 IN BOVINE PRE-IMPLANTATION EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 200
Author(s):  
T. Fujii ◽  
H. Hirayama ◽  
S. Kageyama ◽  
A. Naito ◽  
S. Fukuda ◽  
...  
Keyword(s):  
Real Time ◽  
Cellular Response ◽  
Transcript Level ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Total Rna ◽  
Expression Status

The aquaporins (AQP) are a family of small integral membrane proteins that work as channels for rapid water transport. In mouse embryos, AQP3, AQP7, and AQP9 were found to play important roles in the pre-implantation development or adaptive cellular response to osmotic stress. In addition, because AQP3, AQP7, and AQP9 permeate not only water but also cryoprotectant, such as glycerol, these AQP thought to be involved in the process of cryopreservation. However, little information is available for AQP in bovine embryos. The understanding of the mechanisms mediated by AQP that embryos utilise to survive during culture and the process of cryopreservation will contribute to development of in vitro culture systems and cryopreservation procedures for bovine embryos. The aims of present study were to clarify the expression status of AQP3, AQP7, and AQP9 in bovine pre-implantation embryos, and to evaluate the expression level of AQP3 in in vivo-derived (IVD) and IVF bovine embryos. For production of IVF embryos, cumulus-oocyte complexes (COC) were aspirated from ovaries collected at a local slaughterhouse. The COC were in vitro matured, fertilized, and then cultured for 7 days. The IVD embryos at early blastocyst (EB) and blastocyst (BC) stage were obtained from donor cows treated with superovulation and AI. In experiment 1, in order to clarify the expression of AQP3, AQP7, and AQP9 mRNA in bovine pre-implantation embryos, total RNA was extracted from pools of 30 IVM oocytes, pools of 15 IVF embryos at 2- to 4-cell, 8- to 16-cell, compaction morula (CM), EB, BC, and expanded blastocyst (ExBC) stage, and RT-PCR was performed followed by agarose gel electrophoresis. In experiment 2, in order to clarify the expression status of AQP3, AQP7, and AQP9 during bovine pre-implantation development after zygotic gene activation, total RNA was extracted from pools of 15 IVF embryos at 8- to 16-cell, CM, EB, BC and ExBC stage (n = 5), and relative quantifications of AQP mRNA were performed using real-time RT-PCR. Data were analysed by Scheffé’s method. In experiment 3, AQP3 transcript levels in single IVD and IVF embryos at EB and BC stages (n = 10) was evaluated by real-time RT-PCR. Data were analysed by Mann-Whitney’s U test. In experiment 1, AQP3 and AQP7 transcripts were detected in IVM oocytes and all stages of embryos. AQP9 mRNA was detected in IVM oocytes and in 2- to 4-cell, 8- to 16 cell, CM, and EB stage embryos, but was not detected in BC and ExBC stage embryos. In experiment 2, AQP3 and AQP7 transcript levels were significantly increased from 8- to 16-cell to CM and EB stage, and significantly decreased from EB to BC and ExBC stages (P < 0.05). AQP9 transcript level was significantly decreased from 8- to 16-cell to CM and EB stage (P < 0.05). In experiment 3, AQP3 transcript level in IVF embryos was significantly lower than that in in vivo embryos at the BC stage (P < 0.05). Our results indicate that AQP3 and AQP7 may have specific roles at morula and EB stage in bovine embryos. In addition, AQP3 expression is influenced by developmental condition of bovine embryos.

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