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

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab162 ◽

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.

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166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab166 ◽

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.

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136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab136 ◽

2005 ◽

Vol 17 (2) ◽

pp. 219 ◽

Cited By ~ 5

Author(s):

C.E. Ferguson ◽

T.R. Davidson ◽

M.R.B. Mello ◽

A.S. Lima ◽

D.J. Kesler ◽

...

Keyword(s):

Embryo Development ◽

Direct Effect ◽

Blastocyst Stage ◽

Control Group ◽

Bovine Embryo ◽

Bovine Embryos ◽

Direct Role ◽

Treatment Groups ◽

And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

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83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab83 ◽

2014 ◽

Vol 26 (1) ◽

pp. 155

Author(s):

L. Baldoceda ◽

C. Vigneault ◽

P. Blondin ◽

C. Robert

Keyword(s):

In Vitro Culture ◽

Fluorescent Microscopy ◽

Vitamin K2 ◽

Control Group ◽

Mitochondrial Activity ◽

Bovine Embryo ◽

Bovine Embryos ◽

Blastocyst Rate ◽

Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

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Influence of Injectable Progesterone on the Pregnancy Rate of Heifers Receiving Bovine Embryos

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.104573 ◽

2020 ◽

Vol 48 ◽

Author(s):

Caio Cezar Da Silva ◽

Hélton Aparecido Garcia Gregianini ◽

Jennifer Teodoro Ferreira Gregianini ◽

José Antônio Dell’Aqua Junior ◽

Jefferson Viana Alves Diniz ◽

...

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Physiological State ◽

Bos Indicus ◽

Fixed Time ◽

Treated Group ◽

Pregnancy Rates ◽

Control Group ◽

Bovine Embryos

Background: In vitro embryo production (IVEP) allows the spread of superior animal genetics, but pregnancy rates show a high variability with this biotechnique. In the initial stage of pregnancy, progesterone plays a fundamental role in uterine preparation, acting on embryonic growth, implantation, and development. However, on the day of the IVEP transfer to the recipients, progesterone levels may be lower than that expected, influencing the uterine environment and, consequently, the pregnancy rate. Therefore, the objective of this study was to evaluate the pregnancy rate in heifers after the administration of injectable progesterone (P4) in the fixed-time embryo transfer (FTET) protocol.Materials, Methods & Results: The experiment was conducted inside a rural property near the city of Rio Branco, Acre, Brazil. The experimental group consisted of 232 animals, including 78 zebuine (Bos indicus) and 154 mixed (½ blood B. indicus and ½ blood B. taurus) animals, aged between 16 and 24 months, with a mean weight of 300 and 330 kg for zebuine and mixed animals, respectively. The selected animals were previously synchronized using the progesterone-estrogen-prostaglandin-estrogen protocol. Embryo transfer was performed on day 18 of the protocol, which was 9 days after the removal of intravaginal progesterone implant. On day 15 of the protocol, that is, 144 h (6 days) after the device removal, the animals were randomly distributed into two experimental groups: Control Group (CG; 0.5 mL of 0.9% saline solution, intramuscular) and Treated Group (P4G; 0.5 mL of injectable P4, 150 mg, intramuscular). Chi-square test was used for the statistical analysis of the pregnancy rate at a 5% probability. After 23 days of embryo transfer, pregnancy was diagnosed by ultrasonography. The general pregnancy rate, considering all groups (CG and P4G) and breeds included, was 55.17% (128/232). The pregnancy rates of the P4G and CG groups, regardless of breeds, were 55.08% (65/118) and 55.26% (63/114), respectively, with no statistical difference (P = 0.8344). Angus animals presented a similar pregnancy rate in the P4G and CG groups of 54.93% (39/71) and 57.83% (48/83), respectively. Furthermore, similar results were found for the Nellore breed, with no difference in pregnancy rate between the CG (55.81%, 24/43) and P4G (48.57%, 17/35) groups.Discussion: The strategy of using P4 to increase the pregnancy rate is very widespread in fixed-time artificial insemination (FTAI) protocols, although differences still exist. The literature presents zero or negative effects, as in this study, when exogenous P4 was used to increase endogenous P4 concentrations and, consequently, the pregnancy rate. However, some studies demonstrated the beneficial effects of increasing blood P4 concentrations, and that the increased fertility depends on the method and time of P4 supplementation and the animal’s physiological state. In this context, the fact that the injectable progesterone supplementation did not increase the pregnancy rate in this study is justified mainly by factors such as the animal category used (heifers), time of P4 supplementation (day 4 after ovulation), biotechnology used (FTET), and the heterogeneous characteristics related to phases of the estrous cycle of heifers. Supplementation with 150 mg of injectable long-acting progesterone intramuscularly did not interfere in the pregnancy rate of Angus and Nellore heifers receiving bovine embryos.

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127 LETHAL EFFECT OF HIGH CONCENTRATIONS OF PARECOXIB AND FLUNIXIN MEGLUMINE ON THE IN VITRO CULTURE OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab127 ◽

2009 ◽

Vol 21 (1) ◽

pp. 163 ◽

Cited By ~ 1

Author(s):

E. M. Razza ◽

R. A. Satrapa ◽

C. F. Silva ◽

R. A. L. Simões ◽

T. Nabhan ◽

...

Keyword(s):

Cumulus Cells ◽

Lethal Effect ◽

Control Group ◽

Published Data ◽

Bovine Embryo ◽

Bovine Embryos ◽

Flunixin Meglumine ◽

Plastic Bags ◽

Drug Concentrations

The aim of this experiment was to evaluate the effects of cycloxigenase inhibitor drugs, i.e. flunixin meglumine (FM) and parecoxib (P), on the development of bovine embryos cultured in vitro until the blastocyst and hatched blastocyst stages. Immature oocytes were aspirated from slaughterhouse ovaries and morphologically selected for IVM (Monteiro FM et al. 2007 Anim. Reprod. 4, 51–58). Twenty hours after maturation (39°C and 5% CO2 in air), matured oocytes were transferred to fertilization media, inseminated with frozen–thawed semen, and incubated for 10 to 12 h. Presumptive zygotes (PZ) were then transferred to TCM 199 HEPES medium, vortexed to remove cumulus cells and finally to drops of IVC media (SOFaaci plus 5% BFS [Gibco] with 13 mm sodium pyruvate). Each drop of IVC medium had appropriate concentrations of FM (0.14/n = 123; 1.4/n = 122; 14/n = 117; 140/n = 44 or 1400 μg mL–1/n = 44 PZ) or P (0.09/n = 134; 0.9/n = 109; 9/n = 118; 90/n = 113 or 900 μg mL–1/n = 45 PZ), besides extra drops as control groups (CFM; n = 124 and CP; n = 149 PZ). Based on published data from bovine (FM) and human (P) administered concentrations, it was calculated the blood concentration to a bovine weighing 450 kg (FM = 14 and P = 9 μg mL–1). Both drugs were used from available commercial preparations, and in a pilot test, there were no deleterious effects of the solvent itself on the blastocyst and hatched blastocyst rates. During culture, petri dishes containing PZ/embryos were kept into plastic bags, under controlled atmosphere of 5% O2, 5% CO2 and 90% N2 at 39°C. There were 11 replicates for each treatment. In all drops (both drug concentrations and control group) the blastocyst and hatched blastocyst rates (BR and HBR, respectively) were evaluated at 144 and 192 h after fertilization, respectively. Statistical analysis was performed with ANOVA on ranks (Dunn’s test a posteriori and significance being considered when P < 0.05; BioEstat version 5.0). According to the results, FM (1400 and 140 μg mL–1) and P (900 μg mL–1) concentrations were toxic enough for a complete inhibition of in vitro bovine embryo development. There were no significant differences among the other drug concentrations and their respective control group, on the BR (27.7 ± 3.9; 29.6 ± 3.4 and 29.8% ± 4.8) and HBR (13.5 ± 4.4; 15.6 ± 3.8 and 22.1% ± 5.1), respectively to 0.14; 1.4 and 14 μg mL–1 for FM; on BR (26.0 ± 2.6; 18.2 ± 4.6; 25.8 ± 5.9 and 23.2% ± 4.8) and HBR (14.1 ± 3.3; 10.2 ± 3.3; 16.8 ± 3.8 and 12.0% ± 3.4), respectively to 0.09; 0.9; 9 and 90 μg mL–1 for P; and on BR (35.3 ± 5.2 and 36.5% ± 3.4) and HBR (26.6 ± 4.5 and 19.8% ± 3.6), respectively for CFM and CP. The results suggest that, during in vitro bovine embryo culture, there was no significant toxicity of either drug, with exception of the complete lethal concentrations of 140 and 1400 μg mL–1 (flunixin meglumine) and 900 μg mL–1 (parecoxib) on blastocyst production. Supported by FAPESP – Brazil (MFGN 06/06491-2 and 07/07705-9; EMR 07/04284-2; RAS; CFS and RALS) and CAPES – Brazil.

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Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

10.21203/rs.3.rs-762557/v1 ◽

2021 ◽

Author(s):

Aimé Jazmín Garza Arredondo ◽

Diana Elisa Zamora Ávila ◽

Uziel Castillo Velásquez ◽

Gustavo Moreno Degollado ◽

José Fernando De La Torre Sánchez ◽

...

Keyword(s):

Heat Shock ◽

Blastocyst Stage ◽

Vital Role ◽

Control Group ◽

Bovine Embryo ◽

Bovine Embryos ◽

Embryo Viability ◽

Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

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

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab101 ◽

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.

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210 COMPARISON OF A RECOMBINANT TRYPSIN v. THE PIG PANCREATIC EXTRACT ON IN VITRO-PRODUCED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab210 ◽

2008 ◽

Vol 20 (1) ◽

pp. 184

Author(s):

K. J. Mattson ◽

B. R. Devlin ◽

N. M. Loskutoff

Keyword(s):

Gradient Centrifugation ◽

Bottom Layer ◽

Control Group ◽

Original Research ◽

Bovine Embryo ◽

Bovine Embryos ◽

Ongoing Research ◽

Pathogenic Agents

According to the Manual of the International Embryo Transfer Society, trypsin can be used to remove certain pathogenic agents from in vivo-derived embryos. Research is currently in progress to determine whether trypsin can also remove pathogenic agents from semen. The original research on embryos involved the use of trypsin from pig pancreatic extracts. Because of stricter guidelines from international regulatory agencies on the use of animal products, several recombinant serine protease products are now becoming available. TrypZean (Sigma, St. Louis, MO, USA) is a recombinant developed from corn and is the first bovine sequence recombinant trypsin to contain no animal by-products. As part of our ongoing research on the effects of trypsin on sperm, the goal of this investigation was to examine the development of bovine embryos produced from sperm treated with the recombinant TrypZean compared with pig pancreas trypsin (Sigma) and a control (no trypsin). Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 9 replications. Semen was collected and pooled from Bos taurus and frozen in an egg-yolk cryodiluent (Biladyl, Minitube, Verona, WI). The semen was processed using density gradient centrifugation composed of 1 mL of 30% Percoll (Sigma), layered over 2 mL of 45% Percoll containing either 0.25% TrypZean (n = 972 oocytes), 0.25% trypsin (n = 1040 oocytes), or no trypsin for the control group (n = 1024 oocytes). The bottom layer for the 2 treatments and control was 2 mL of 90% Percoll containing 10 µg mL–1 of soy-based protease inhibitor (Sigma). The density gradients were centrifuged at 700g for 30 min, after which time the pellets were washed in 5 mL of prewarmed TL HEPES Solution (Cambrex) and centrifuged at 300g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 � 106 sperm mL–1 for in vitro insemination. The results were compared using one-way ANOVA. There were no statistically significant differences (P > 0.05) between any of the measures of embryonic development for the control and either of the treatment groups. Cleavage rates were measured for TrypZean (n = 689, 70.9%), trypsin (n = 729, 70.1%), and the control (n = 757, 73.9%) groups. More embryos reached the morula to blastocyst stages with the TrypZean (n = 367, 53.3%) and trypsin (n = 389, 53.4%) groups than the control (n = 369, 48.7%) group; however, these differences also were not statistically significant (P = 0.91) because of the large variation within the groups. In conclusion, the TrypZean and pig pancreas trypsin treatments of sperm prior to insemination showed no detrimental effects on IVF-derived bovine embryo development.

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