Intact Zona Pellucida Research Articles

Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles.

This study aimed to investigate the effects of different concentrations of N-acetylcysteine (NAC) on the growth, antrum formation, viability, and ultrastructure of bovine secondary follicles cultured in vitro for 18 days. To this end, the follicles were cultured in TCM-199+ medium alone or supplemented with 1.0, 5.0, or 25.0 mM NAC. Follicular growth, antrum formation, viability (calcein-AM and ethidium homodimer-1) and ultrastructure were evaluated at the end of culture period. The results showed that 1.0 mM NAC increased the percentage of growing follicles and the fluorescence intensity for calcein-AM when compared to other treatments (p < 0.05). On the other hand, follicles cultured with 25.0 mM NAC had higher fluorescence intensity for ethidium homodimer-1, which is a sign of degeneration. Ultrastructural analysis showed that oocytes from follicles cultured in control medium alone or with 1 mM NAC had intact zonae pellucidae in close association with oolemmae, but the ooplasm showed mitochondria with a reduced number of cristae. On the other hand, oocytes from follicles cultured with 5 or 25 mM NAC had extremely vacuolated cytoplasm and no recognizable organelles. In conclusion, 1 mM NAC increases cytoplasmic calcein staining and the growth rate in bovine secondary follicles cultured in vitro, but the presence of 5 or 25 mM NAC causes damage in cellular membranes and organelles, as well as reducing the percentages of growing follicles.

Risk of pathogenic virus transmission by somatic cell nuclear transfer: implications for xenotransplantation.

Using somatic cell nuclear transfer for the generation of cloned and transgenic animals bears the risk of transmission of viruses, either by the oocyte or by the introduced donor cell. There is evidence that the zona pellucida (ZP) surrounding the oocyte prevents virus infection; however, virus infections despite intact ZP were reported. Furthermore, the protective ZP has to be penetrated to place the somatic cell in the oocyte's perivitelline space during SCNT. Transmission of viruses also represents a severe problem during in vitro fertilization (IVF). Genetically modified and IVF-produced pigs serve as an important biomedical model for numerous diseases and it is important to evaluate whether infections of the model animals can falsify the research data. Of special significance is this topic in the case of xenotransplantation using genetically modified pigs as donor animals, because transmission of porcine viruses may be harmful to the human recipient. This was repeatedly demonstrated in preclinical pig to non-human primate trials. Therefore, donor pigs, oocytes used for SCNT, and genetically modified donor cells should be screened for potentially zoonotic viruses when creating genetically modified pigs designed for xenotransplantation.

Vitamin E supplementation may negatively affect preimplantation development and mitochondrial ultrastructure of vitrified murine embryos

Objective: To observe the effects of vitamin E on post-vitrification preimplantation development, gross morphology as well as mitochondrial distribution and ultrastructure. Methods: Twenty-four female C57BL/6NTac mice, aged 12-16 weeks, were randomly divided into four groups. Group A did not receive any treatment and served as the control group. Group B was treated with corn oil stripped of tocopherols and served as the vehicle group. Group C was treated with 60 mg/kg body weight of tocotrienol-rich-fraction with corn oil stripped of tocopherols. Group D was treated with 60 mg/kg body weight of alpha-tocopherol with corn oil stripped of tocopherols. All treatments were administered orally for 7 consecutive days. After superovulation and mating with fertile males, 2-cell stage embryos were harvested for vitrification. Post vitrification development in vitro, gross morphology and ultrastructure were compared between groups. Results: The number of 2 and 8-cell embryo, and blastocysts in the treatment and control groups were not significantly different (P>0.05). Following vitrification, all 2-cell embryos had equal-sized blastomeres and intact zona pellucida. Mitochondrial aggregation toward the perinuclear region was seen in all of the treatment groups. Both groups C and D had vacuolated mitochondria, which was reflected in the trend of preimplantation development reduction. Conclusions: Vitamin E supplementation of 60 mg/kg body weight does not improve the viability of healthy embryos according to this study. As a result, the most effective dose of vitamin E supplementation may be determined by the initial quality of the embryos.

Manipulatingearly pig embryos

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a longterm preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demiembryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been bom upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and < 10% develop to morulae or blastocysts in vitro. Pig oocytes can be stimulated parthenogenetically and up to 10% grow to blastocysts in the in vitro culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind considerable amounts of foreign DNA preferably at the post-acrosomal region, suggesting that transgenic animals can be obtained more efficiently than with the usual microinjection procedure. To increase gene transfer efficiency, considerable research efforts have been made to establish embryonic stem (ES) cells, but so far there is no definite proof of totipotency of the generated pig ESlike cells through viable chimaeras. In general, biotechnological procedures are much less advanced in pigs than in cows.

Cryopreservation and IVF in the time of Covid-19: what is the best good tissue practice (GTP)?

Examine good tissue practices as relates to in vitro fertilization, biopsying, and vitrificationto compare current knowledge of ova, sperm, and embryos as vectors for disease transmission as it relates to our current knowledge regarding the SARS-CoV-2 virus.Unknown risks relating to the SARS-CoV-2 virus and sperm, ova, and embryos necessitate a reexamining of how human IVF is performed. Over the last decade, improvements in cryosurvival and live birth outcomes have been associated with zona pellucida breaching procedures (e.g., blastocyst collapsing and biopsying). In turn, today embryos are generally no longer protected by an intact zona pellucida when vitrified and in cryostorage. Additionally, high security storage containers have proven to be resilient to potential cross-contamination and reliable for routine human sperm freezing and embryo vitrification.Several options to current IVF practices are presented that can effectively mitigate the risks of cross-contamination and infection due to the current Covid-19 pandemic or other viral exposures. The question remains; is heightened security and change warranted where the risks of disease transmission likely remain negligible?

Comet assay on one- and two-cell mouse embryos

DNA damage quantified as the comet tail length was assessed using in vitro and in vivo comet assay on one- and two-cell mouse embryos obtained by natural mating. The use of a protocol with three layers of agarose reduces the embryo loss and makes it possible to study a small number of embryos. A significantly lower level of basal, but not induced DNA damage was found in embryos with cleaved zona pellucida compared to embryos with intact zona pellucida. There were no significant differences in the length of the comet's tail between embryos lysed in different lysis solutions, both in cases of basal and induced DNA damage. A significant increase in the comet tail length was detected in one-cell embryos of mice treated with methyl methanesulfonate and etoposide compared to the control. The data show that DNA damage induced in maternal germ cells persists, which can be detected in embryos using the comet assay.

22 The zona pellucida is required for normal development of invitro-produced cat embryos

Limited information have been reported regarding to the role of the zona pellucida during embryo development in the domestic cat. In the present research we compared invitro and invivo development of cat embryos generated with versus without the zona pellucida. Embryos were produced by IVF and cultured up to blastocyst stage accordingly to experimental group: 1) with intact zona pellucida (ZI) and 2) without zona pellucida (ZF). Ovaries of domestic cats were collected by ovariohysterectomy and cumulus-oocyte complexes (COCs) were recovered by slicing. For IVM, COCs were cultured in supplemented medium-199 for 26-28h in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C. For IVF, 1.5-2.5×106 epididymal spermatozoa/mL were incubated with 20-30 COCs in supplemented Tyrode's albumin lactate pyruvate medium for 18h in a 5% CO2 atmosphere at 38.5°C. The zona pellucida of the presumed zygotes was removed by 2-4min of incubation in 2mgmL−1 pronase. In ZI and ZF groups, embryo culture was done in 4-well dishes; in the ZF group the well of the well system was used. The IVC was done in supplemented synthetic oviductal fluid medium, in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C for 8 days. The frequencies of cleavage and development to the morula and blastocyst stages were determined. The relative expression of the pluripotency markers OCT4, SOX2, and NANOG was evaluated by RT-qPCR in the blastocysts using the standard curve method. The SDHA gene was used as internal control. Additionally, the invivo development was evaluated. For this, cat recipients were synchronized with 200IU equine chorionic gonadotrophin followed by 100IU human chorionic gonadotrophin 4 days later, and embryo transfers (ET) were made by mid-ventral laparotomy. The Wilcoxon nonparametric test was used to evaluate the developmental competence and the gene expression analysis. Nine and six replicates were performed in the ZI and ZF groups, respectively. No differences were observed between the ZI and ZF groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morulae rate: 115/155 (74.2%) and 68/116 (58.6%), and blastocysts rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P&amp;gt;0.05). No differences were observed in the expression of OCT4 (P&amp;gt;0.05). However, the expression of SOX2 and NANOG was lower in blastocysts from the ZF group than in those from the ZI group (P&amp;lt;0.05). Finally, three ET were done at the blastocyst stage in both groups. In the ZI group, 8-16 Day 7 blastocysts were transferred per cat, two pregnancies were obtained with one gestational sac in each cat (2/3, 66.6%), and one live kitten was born at Day 64 of pregnancy (1/3, 33.3%). In the ZF group, 7-14 were transferred per cat, and no pregnancies were obtained after ET. In conclusion, in the domestic cat, zona pellucida removal at the presumed zygote stage did not affect invitro development to blastocyst but affect negatively the expression of SOX2 and NANOG. However, the low survival after ET of ZI embryos is a possible indicator that embryo quality might be affecting survival rates. Nonetheless, these results add credence to previous ET studies that provide indirect evidence of a crucial role of the zona pellucida for successful implantation of cat embryos.

Correction: Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection

[This corrects the article DOI: 10.1371/journal.pone.0218963.].

Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.

Anethole improves blastocysts rates together with antioxidant capacity when added during bovine embryo culture rather than in the in vitro maturation medium.

We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

Effect of supplementation of vitrification solution on post-thaw survivability rate of porcine follicular oocytes

Oocytes (510) with two or more cumulus cell layers adhered to zona pellucida obtained by aspiration from follicles (2–8 mm dia) of 127 abattoir porcine ovaries were utilized to study the effect of supplementation of 0.5 M sucrose (Suc), 50 mg/ml polyvinyl pyrrolidone (PVP) and Suc + PVP into two different vitrification solutions, i.e. 35% concentration of both ethylene glycol (EG)+ propylene glycol (PG) and EG + dimethyl sulphoxide (DMSO). Vitrified post-thaw oocytes with intact zona pellucida and vitelline membrane, normal spherical shape and dark and evenly granulated cytoplasm under a stereo-zoom microscope were considered as viable. Out of the three supplements treatments, supplementation with sucrose + PVP combination and sucrose alone resulted in significantly higher mean post-thaw survivability rate of vitrified oocytes both for EG + PG and EG + DMSO as compared to the supplementation with PVP alone. It was concluded that addition of sucrose alone or in combination with PVP in vitrification solution, significantly improved survivability of vitrified porcine follicular oocytes.

Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts.

The introduction of exogenous molecules into embryos is required for analyses of molecular dynamics and specific gene functions during early embryonic development. Electroporation is an effective method to transport exogenous molecules into cells, but is rarely used in bovine embryos. First, we evaluated the viability of in vivo-derived bovine blastocysts after electroporation with fluorescein (FAM) labeled-oligonucleotides with varying pulse numbers (3, 5, 7, and 10), while keeping the pulse duration at 1 msec and the electric field of 20 V/mm. Next, we examined the effects of zona pellucida status on blastocyst quality after electroporation, by comparing the average diameter of blastocysts before and after electroporation using blastocysts with intact zona pellucida and hatching/hatched blastocysts. Electroporation successfully introduced exogenous molecules into in vivo-derived bovine blastocysts without loss of viability. Moreover, the status of the zona pellucida may be associated with the quality of blastocysts after electroporation.

CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice.

Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos' genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.

Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1)

ABSTRACT Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P&lt; 0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.

Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol.The experimentation was made twice. For each replicate 100 (8–16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO2, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos 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. Each batch of washed embryos and each wash pellets were tested using PCR.C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ± 9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ± 0.19 × 103 bacteria/mL).These results demonstrate that C. abortus adheres to 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 fails to remove it.

Effect of type and concentration of cryoprotectant on post-thaw survivability of vitrified porcine follicular oocytes

A total of 950 and 510 oocytes with two or more cumulus cell layers adhered to zona pellucida obtained by aspiration from follicles (2–8 mm dia) of 315 and 135 abattoir porcine ovaries, respectively, were utilized in the study to find the effect of cryoprotectant and concentration on post-thaw survivability of porcine follicular oocytes. Vitrified post-thaw oocytes with intact zona pellucida and vitelline membrane, normal spherical shape and dark and evenly granulated cytoplasm under a stereo-zoom microscope were considered as viable. Out of the 10 cryoprotectant treatments i.e. ethylene glycol (EG), propylene glycol (PG), dimethyl sulphoxide (DMSO), glycerol (GL), EG + PG, EG + DMSO, EG + GL, PG + DMSO, PG + GL and DMSO + GL used for vitrification of oocytes at a concentration of 35%, the post-thaw survivability rate was the highest in EG +DMSO followed by EG + PG group which had significantly higher mean post-thaw survived oocytes as compared to GL, PG + DMSO, PG + GL and DMSO + GL groups. Exposing the oocytes to 30, 35 and 40% of EG + DMSO and EG + PG yielded no significant difference in post-thaw survivability rate of vitrified oocytes, although the highest value was obtained with 35%. It was concluded that 35 to 40% EG + DMSO yielded efficient vitrification of porcine oocytes.

90 Risk of Coxiella burnetii Transmission via Embryo Transfer Using In Vitro Early Caprine Embryos

Previous experiments using in vitro infection have shown that Coxiella burnetii has a strong tendency to adhere to the zona pellucida (ZP) of in vivo-derived goat embryos, and the washing procedure recommended by the International Embryo Technology Society (IETS) for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology 80, 571-575). The aim of this study was, for in vitro-produced caprine embryos infected in vitro, to (1) evaluate the ability of C. burnetii to adhere to intact ZP, (2) test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii, and (3) determine, by confocal microscopy, the bacteria location. One hundred ZP-intact caprine embryos, produced in vitro, at the 8- to 16-cell stage were randomly allocated into 11 batches of 8 to 9 embryos. Nine batches were incubated for 18 h with 109 Coxiella/mL of CbB1 strain (ISP, INRA, Val de Loire, France). The embryos were then recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, 2 batches of embryos were subjected to similar procedures but without exposure to C. burnetii to serve as the control group. One of the 9 batches of infected embryos and 1 of the 2 non-infected control batches were used to perform immunolabelling to localise the bacteria. Coxiella burnetii DNA was detected by conventional PCR in all 8 batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryos of the control group. The first 5 washing media of the infected groups were consistently positive and Coxiella DNA was detected up to the tenth wash in 2 batches. After immunolabelling, the observation of embryos under confocal microscopy allowed us to localise C. burnetti on the external part of the ZP without deep penetration. The presence of C. burnetii was seen on the surface of the ZP, with bacterial loads differing from one embryo to another in the same batch. This study clearly demonstrates that C. burnetii, after in vitro infection at 109 Coxiella mL−1, stick strongly to the external part of the ZP of in vitro-produced early caprine embryos without profound penetration. The 10-washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria in caprine embryos. Nevertheless, the finding of C. burnetii DNA by conventional PCR does not imply that the bacteria found remain infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of caprine embryos infected by C. burnetii would eliminate or inactivate the bacteria from the ZP of in vitro-produced goat embryos.

Caprine arthritis encephalitis: an example of risk assessment for embryo trading.

The risk of transmission of caprine arthritis encephalitis virus (CAEV) during embryo transfer has been demonstrated in vivo through the detection of CAEV proviral DNA in: (1) flushing media for embryo collection; (2) cells of the cumulus oophorus surrounding the oocytes, ovarian follicle, oviduct and uterine tissues; and (3) testis, epididymis, vas deferens and vesicular glands. Experimentally infected embryos without a zona pellucida (ZP), washed 10 times with Minimum Essential Media (MEM) and 5% Fetal Calf Serum (FCS) solution, were capable of transmitting CAEV. In vitro we demonstrated that granulosa, oviductal, epididymal and embryo cells are fully susceptible to CAEV infection and allow active replication. However, AI with in vitro-infected semen can result in the production, after ten washing, of CAEV-free embryos, and ten washing in vitro- or in vivo-infected embryos with an intact ZP, or ten washing oocytes with an intact ZP, resulted in the production of virus-free female gametes or embryos that can be used for IVF or embryo transfer. Therefore, we have demonstrated that: (1) that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV; and (2) embryo transfer can be used under field conditions to produce CAEV-free kids from CAEV-infected biological mothers.

28 IMPROVED HATCH RATE AFTER PARTIAL DISSECTION OF ZONA PELLUCIDA IN CLONED PIG EMBRYO

For last 20 years, the efficiency of animal cloning has remained extremely low, despite many attempts to improve it. Although nuclear transfer experiments have been almost optimized, artificial holes are inevitably made in the zona pellucida (ZP) during nuclear transfer experiments, such as enucleation of maternal genome or injection of nuclear donor. Hatching from the ZP is a prerequisite for mammalian embryo implantation, and the condition of the ZP has a lot of influence on hatching. The present studies were performed to investigate the effects of artificial holes in the ZP, because of the nuclear transfer procedure, on hatching of clone embryos in pigs. All experiments were done in triplicate. Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). First, we made a slit in the ZP of porcine parthenote that was identical to the artificial holes of nuclear transfer experiment and compared in vitro development of Day 7 embryos with control group with intact ZP. Of slit blastocysts, 89.9% (80/89) were trapped at a slit, which looked like typical figure-eight shape, and did not complete the hatching process until Day 8, though 68.8% (64/93) of control blastocysts completed the hatching at Day 7. Then, to solve these abnormal hatchings caused by a slit in the ZP, we applied partial zona dissection (PZD) to porcine clone embryos and compared the hatching process with that of conventional clone embryos. Contrary to conventional clone blastocysts that were trapped at slit in the ZP (91.4%; 43/47), 89.5% (60/67) of clone blastocysts in PZD group were preferentially hatched through dissected hole at Day 7. These results suggest that trapping of conventional clone blastocyst in a slit of the ZP could be avoided by PZD. Through this study, we demonstrated that a slit in the ZP would hinder a blastocyst from hatching from the ZP and that partial dissection at the ZP could help clone blastocyst to hatch without trapping at a slit in the ZP. This assisted hatching in clone embryos would be useful for the successful hatching of clone blastocysts with a capacity of full-term development, so that the efficiency of animal cloning might be improved.