166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?

2013 ◽  
Vol 25 (1) ◽  
pp. 231
Author(s):  
A. Alsaleh ◽  
J. L. Pellerin ◽  
C. Roux ◽  
M. Larrat ◽  
G. Chatagnon ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Early Embryo ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Tissue Samples ◽  
Worldwide Distribution

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Detection of significant bacterial loads in flushing media and tissue samples (oviducts and uterine horns) from the genital tracts of nonpregnant goats is a risk factor for in utero infection and transmission during embryo transfer (Alsaleh et al. 2011 CIMID 34, 355–360). The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the washing protocol recommend by the IETS for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and PCR, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9–9, 11–11, and 4–4 in replicates 1, 2, and 3, respectively) were placed in 1 mL of MEM containing 107 C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37°C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3–3, 5–5, and 2–2 in replicates 1, 2, and 3, respectively) were submitted to the same procedures but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 h at 13 000g. The washed embryos and pellets were tested by PCR. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first 5 washing fluids for ZP-free embryos and in the first 8 washing fluids for ZP-intact embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly demonstrate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to cling to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

101 RISK OF COXIELLA BURNETII TRANSMISSION BY EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 165
Author(s):  
A. Alsaleh ◽  
J. L. Pellerin ◽  
D. M. Garcia ◽  
D. Tainturier ◽  
F. Fieni
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Economic Losses ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Worldwide Distribution ◽  
Embryo Cells ◽  
Means Of Transmission

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main sources of infection for humans. In cattle, infection is frequently asymptomatic, but it may cause abortion, reproductive failure (metritis, placentitis, and infertility), and economic losses. A previous study in goats showed that Coxiella burnetii had a strong tendency to cling to the zona pellucida (ZP) after in vitro infection and the washing procedure recommended by IETS for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology). The aims of this study were to determine (1) whether Coxiella burnetii would adhere to the intact ZP (ZP-intact) of early in vitro-produced bovine embryos, (2) whether the bacteria would adhere to or infect the embryo cells (ZP-free) after in vitro infection, and (3) the efficiency of the washing protocol recommended by the IETS. One hundred and sixty 8- to 16-cell bovine embryos produced in vitro were randomly divided into 16 batches of 10 embryos each. Twelve batches (8 ZP-intact and 4 ZP-free) were incubated in medium containing C. burnetii CbB1 (IASP, INRA Tours, France). After 18 h of incubation at 37°C and 5% CO2 in air, the embryos were washed in 10 successive baths of a phosphate buffer saline (PBS) and 5% FCS solution in accordance with the IETS guidelines. In parallel, 4 batches (2 ZP-intact and 2 ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. The 10 washing fluids for all batches were collected and centrifuged for 1 h at 13 000 × g. Embryo and pellet washing were tested by C-PCR. Coxiella burnetii DNA was found in all ZP-intact and ZP-free embryo batches after 10 successive washes. It was also detected in the first 4 washing fluids for ZP-intact embryos and in the 10th washing fluid for 2 of the 4 batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adhere and (or) penetrate the early embryonic cells as well as the ZP of in vitro bovine embryos after in vitro infection and the standard washing protocol recommended by the IETS for bovine embryos 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, or both. Further studies are needed to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

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.

162 CAN CHLAMYDIA ABORTUS BE TRANSMITTED BY EMBRYO TRANSFER IN GOATS?

2015 ◽  
Vol 27 (1) ◽  
pp. 172
Author(s):  
J. L. Pellerin ◽  
A. Ashraf ◽  
M. Oseikria ◽  
K. Laroucau ◽  
F. Vorimore ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Chlamydia Abortus ◽  
Zoonotic Risk ◽  
Embryo Cells ◽  
Means Of Transmission ◽  
Infectious Form

Chlamydia abortus is a gram-negative obligate intracellular bacterium. Its lifecycle includes a resistant infectious form and a metabolically active non-infectious form. Chlamydia abortus infection results in abortion in goats; in nonpregnant animals the infection is usually subclinical. Chlamydia abortus presents a major zoonotic risk for pregnant women. The aim of this study was to investigate whether the embryonic zona pellucida (ZP) protects early embryo cells from infection and to test the efficacy of the washing protocol recommended by the IETS for bovine embryos. The study was performed in triple replicate: 14 donor goats, certified negative by ELISA and PCR to C. abortus, were synchronized, superovulated, and subsequently inseminated by males controlled negative for C. abortus. Fifty-two ZP-intact embryos (8–16 cells) were collected 4 days later, by laparotomy. The embryos were randomly divided into 12 batches. Nine batches of 5 embryos were incubated in a medium containing 4 × 107 Chlamydia mL–1, AB7 strain. 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 PBS and 5% FCS solution in accordance with IETS guidelines for bovine embryos. In parallel, 3 batches of ZP-intact embryos (2, 2, and 3 embryos in the first, second, and third batches, respectively) were used as controls by being subjected to similar procedures, but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 h at 13 000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at –20°C before examination for evidence of C. abortus using RT-PCR. Chlamydia abortus DNA was found in all batches of infected ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the tenth wash fluid for 4 batches (4/9) of infected embryos. As expected, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos 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 goat to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of infected caprine embryos can eliminate C. abortus from the ZP.

scholarly journals The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cody B. Smith ◽  
Charles Evavold ◽  
Gilbert J. Kersh
Keyword(s):  
Coxiella Burnetii ◽  
Antimicrobial Agents ◽  
Q Fever ◽  
Antimicrobial Treatment ◽  
Etiologic Agent ◽  
Growth And Survival ◽  
Intracellular Compartments ◽  
The Impact

AbstractCoxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

scholarly journals Coxiella burnetii Interaction with Neutrophils and MacrophagesIn Vitroand in SCID Mice following Aerosol Infection

2013 ◽  
Vol 81 (12) ◽  
pp. 4604-4614 ◽  
Author(s):  
Alexandra Elliott ◽  
Ying Peng ◽  
Guoquan Zhang
Keyword(s):  
Immune Response ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Natural Infection ◽  
Scid Mice ◽  
Innate Immune ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium that causes acute and chronic Q fever in humans. Human Q fever is mainly transmitted by aerosol infection. However, there is a fundamental gap in the knowledge regarding the mechanisms of pulmonary immunity againstC. burnetiiinfection. This study focused on understanding the interaction betweenC. burnetiiand innate immune cellsin vitroandin vivo. Both virulentC. burnetiiNine Mile phase I (NMI) and avirulent Nine Mile phase II (NMII) were able to infect neutrophils, while the infection rates were lower than 29%, suggesting thatC. burnetiican infect neutrophils, but infection is limited. Interestingly,C. burnetiiinside neutrophils can infect and replicate within macrophages, suggesting that neutrophils cannot killC. burnetiiandC. burnetiimay be using infection of neutrophils as an evasive strategy to infect macrophages. To elucidate the mechanisms of the innate immune response toC. burnetiinatural infection, SCID mice were exposed to aerosolizedC. burnetii. Surprisingly, neutrophil influx into the lungs was delayed until day 7 postinfection in both NMI- and NMII-infected mice. This result suggests that neutrophils may play a unique role in the early immune response against aerosolizedC. burnetii. Studying the interaction betweenC. burnetiiand the innate immune system can provide a model system for understanding how the bacteria evade early immune responses to cause infection.

204 RELATIVE MESSENGER RNA ABUNDANCE OF HYALURONAN RECEPTORS AND SYNTHASES ON IN VITRO- AND IN VIVO-DERIVED DAY 7 AND DAY 13 BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
M. Clemente ◽  
A. T. Palasz ◽  
J. de La Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Messenger Rna ◽  
Developmental Stages ◽  
Transcript Abundance ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cd44 Receptor ◽  
Mrna Extraction

Hyaluronan (HA), which progressively increases during embryogenesis, is a glycosaminoglycan that plays a major role in oocyte/embryo development (Fenderson et al. 1993 Differentiation 54, 85–95). One of the main functions of HA is to participate in the cell proliferation and migration that are controlled by HA receptors, RHAMM and C44, and by the presence of different HA synthases, Has1, Has2, and Has3. All have very distinctive features and functions at different stages of embryo development. The objective of this study was to determine the relative mRNA abundance of HA receptors and synthases in Day 7 and 13 bovine embryos derived in vitro or in vivo. In vitro embryos were produced by standard oocyte maturation and fertilization procedures. Presumptive zygotes were cultured in groups of 25 in 25-μL droplets of synthetic oviduct fluid supplemented with 5% FCS at 39°C, 5% CO2, and 5%O2 with maximum humidity. In vivo blastocysts were collected from superovulated heifers on Day 7 (estrus = Day 0) by uterine flushing and on Day 13 immediately after slaughter by flushing the dissected reproductive tracts. All embryos were frozen in LN2 and stored at –80°C for mRNA extraction. Quantification of transcripts for RHAMM and CD44 receptors and Has2 and Has3 synthases was performed on groups of ten Day 7 blastocysts (3 groups for in vivo or in vitro) and individual Day 13 embryos (7 embryos in vivo or in vitro) by real-time quantitative RT-PCR. Data on differences in transcript abundance were analyzed by ANOVA. The relative abundance of the Has2 and Has3 synthases was similar between in vivo and in vitro embryos, irrespective of their developmental stage. The quantity of CD44 was significantly higher in in vitro compared with in vivo embryos only on Day 7. However, the quantity of RHAMM receptor was higher on Day 13 in in vitro compared with in vivo embryos. When the comparison was done between developmental stages (Day 7 v. Day 13) for in vivo and in vitro embryos, we found that in vivo-produced Day 7 blastocysts expressed significantly more RHAMM receptor than embryos on Day 13. The reverse pattern of expression was shown for CD44 receptor. For in vitro embryos, the only difference observed was for Has3, which was up-regulated on Day 13 compared with Day 7 embryos. In conclusion, the present study demonstrates, for the first time, developmental changes in the abundance of RHAMM and CD44 receptor mRNA and Has2 and Has3 synthase mRNA in in vivo and in vitro bovine-derived embryos on Day 7 and 13. We believe that our results will provide new insight into the potential role of this intriguing multifunctional molecule in bovine early embryo development.

Risk of Coxiella burnetii transmission via embryo transfer using in vitro early bovine embryos

2014 ◽  
Vol 81 (6) ◽  
pp. 849-853 ◽  
Author(s):  
A. Alsaleh ◽  
F. Fieni ◽  
D. Moreno ◽  
E. Rousset ◽  
D. Tainturier ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Bovine Embryos

scholarly journals 150 ENVIRONMENT OF THE EARLY EMBRYO AND ITS EFFECT ON DEVELOPMENT AND POSTNATAL LIFE

2005 ◽  
Vol 17 (2) ◽  
pp. 225
Author(s):  
A. Watkins ◽  
A. Wilkins ◽  
T. Papenbrock ◽  
C. Osmond ◽  
M. Hanson ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Embryo Transfer ◽  
Litter Size ◽  
Early Embryo ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Elevated Systolic Blood Pressure

We have investigated the impact of mouse early embryo in vitro culture environment on (a) short-term blastocyst development and (b) long-term postnatal growth and physiology after embryo transfer. In vitro-developed blastocysts, cultured from the 2-cell stage, had reduced inner cell mass (ICM) and trophectoderm (TE) cell numbers when compared to in vivo-derived blastocysts at 96 h post-hCG (n = 13–39, P < 0.05). Despite the retardation in blastocyst development, the ICM:TE ratio was equivalent in both treatment groups. Using embryo transfer techniques, we compared the postnatal development of embryos cultured in vitro from the 2-cell to the blastocyst stage (termed “in vitro” mice) with offspring generated from blastocysts developed in vivo, but which also underwent embryo transfer (termed “in vivo” mice). These two treatment groups were in turn compared with mice derived from naturally mated mothers, which had their mean litter size at birth adjusted to a size comparable with that of the in vitro and in vivo mice (a mean of 6 animals) and which had not been transferred. All data were analyzed using a multilevel random effects regression model which took into account between-mother and within-mother variation in litter size for parameters measured from individual animals. No significant differences in birth weight were observed between in vitro and in vivo offspring. However, in vitro offspring were significantly lighter than in vivo offspring in a gender-dependent manner at 2 weeks of age (males, P = 0.009) and at 6 and 11 weeks of age (females, P = 0.037 and 0.035, respectively). In addition, at 4 weeks of age, the in vivo males became significantly lighter when compared to the naturally mated males (P = 0.034). At 8 weeks of age, the in vivo females had a significantly elevated systolic blood pressure when compared to the in vitro females (P = 0.003); however, at 21 weeks of age, both in vitro males and females had a significantly elevated blood pressure when compared to in vivo offspring (P < 0.003). At 8, 15, and 21 weeks of age, offspring derived from transferred embryos developed with significantly elevated systolic blood pressure when compared to non-embryo transfer offspring (P < 0.05). No significant differences in serum angiotensin-converting enzyme activity (a potent regulator of systolic blood pressure) was observed between the treatment groups. Significantly altered liver:body weight ratios were observed between the in vitro and in vivo males, and between the in vitro and the naturally mated (6) females (P < 0.038). All of the above data are independent of litter size. These data support the hypothesis that early embryo environment can influence postnatal growth and cardiovascular physiology. This research was funded by an MRC research grant to TPF, and by a DOHaD studentship.

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?

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