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PubMed2006

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos.

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.

Animals↗

PubMed1997

The role of albumin in the release of platelet-activating factor by mouse preimplantation embryos in vitro.

Platelet-activating factor (PAF) produced by embryos remained associated with mouse four-cell embryos after culture in vitro and was also released into the medium. The release of PAF into medium required albumin as a media supplement and the amount of PAF released increased (P < 0.05) with increasing albumin concentration. There was a trend for the amount of PAF remaining associated with embryos to decrease as the extracellular albumin concentration increased. The association of released PAF with albumin was confirmed by size fractionation with size exclusion membranes and high performance gel filtration, and by affinity chromatography (Cibacron blue and anti-BSA) and native PAGE. PAF released from embryos was not degraded by serum PAF:acetylhydrolase (PAF:AH; E.C. 3.1.1.47) after exposure for 24 h to the serum in vitro, while an equivalent concentration of synthetic PAF added to identical media was readily degraded, suggesting that PAF released from the embryo was protected from PAF:AH action. However, when the medium was subjected to organic extraction by the Bligh-Dyer (methanol/chloroform) method and the resulting extract added to equivalent media, embryo-derived PAF was readily degraded by PAF:AH. Furthermore, PAF in embryo-conditioned medium (30 two-cell embryos for 24 h) could not be detected after direct assay of the culture medium by radioimmunoassay or bioassay (platelet aggregation in vitro), yet after extraction, purification and addition to medium with BSA, the embryo-derived PAF was readily detected in either assay. To determine whether the different behaviour of synthetic PAF and embryo-derived PAF resulted from differences in the nature of their binding to albumin, the location to which PAF bound was assessed by limited proteolytic digestion of albumin. Digestion with pepsin or trypsin showed that embryo-derived PAF was located exclusively between amino acids 240 and 386 (domain II) of albumin. Most synthetic PAF added to equivalent medium not exposed to embryos was not at this location, suggesting that PAF released from embryos bound to a site on albumin not generally accessible to synthetic PAF added to similar media.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1995

Nuclear transfer in the bovine using microinjected donor embryos: assessment of development and deoxyribonucleic acid detection frequency.

Bovine embryos that had been microinjected with DNA were examined for their potential use as donor embryos in nuclear transfer. Donor embryos were obtained from oocytes collected by transvaginal oocyte aspiration, matured and fertilized in vitro, microinjected with a murine whey acidic protein-human protein C genomic DNA construct, and cultured in vitro on liver cells of buffalo rat (Rattus norvegicus). Blastomeres from these embryos were transferred into enucleated bovine oocytes received from an abattoir by electrofusion at 40 h postmaturation. Following 7 d of culture, the developmental stage was recorded, and resulting embryos were prepared for analysis by polymerase chain reaction. Embryos that were derived from microinjected donor embryos did not differ from control donor embryos (11 vs. 8.6%) in development to the morula and blastocyst stage. Of the biopsies from 20 microinjected donor embryos, 19 were positive for the injected DNA. Of 37 embryos developing normally, only 12 (32.4%) were positive for the injected DNA. These results indicate that microinjected embryos can be successfully used in a nuclear transfer program to produce additional viable embryos and that these embryos may be reliably screened for the transgene for transfer to recipients.

Animals↗

PubMed2006

[Embryo quality evaluation according to the speed of the first cleavage after IntraCytoplasmic Sperm Injection (ICSI)].

OBJECTIVE: To evaluate morphological parameters of embryos obtained in the process of ICSI. DESIGN: A prospective study. SETTING: Centre of Assisted Reproduction, Dept. of Obstetrics and Gynecology, Palacký University Medical School, Olomouc. METHODS: In the present study 1116 embryos developing after ICSI (IntraCytoplasmic Sperm Injection) procedure in the period of 2001-2004 were evaluated. The beginning of the mitotic cleavage was assessed within the interval of 22-27 hours after insemination. The embryos were divided into three groups according to the speed of their division as Early Cleavage (EC) embryos, where two blastomeres were present at the time of assessment, Break Down ProNuclei stage (BDPN) where the pronuclei had already disappeared, and ProNuclei (PN) embryos, where both pronuclei were still present. In these groups the degree of fragmentation was evaluated on day two of cultivation and embryos were divided into four categories as: A--regular blastomeres, without fragmentation, B--irregular blastomeres or fragmentation below 30%, C--fragmentation 30-50%, D--fragmentation above 50%. The speed of further cleavage and average number of blastomeres were evaluated on day two and three of cultivation. Statistical analysis was preformed at the Palacky University Computer Centre. The chi2 test and t-test for independent samples were used. RESULTS: EC embryos were found in 37.4%, BDPN in 33.1% and PN in 29.5%. The degree of fragmentation between evaluated groups of embryos were statistically significant (p = 0.000). EC embryos were less fragmented (p = 0.000), had more blastomeres at the time of evaluation (p = 0.000) and their speed of cleavage was faster (p = 0.000). The cleavage of EC embryos was faster in comparison with the PN group (p = 0.000), but there were no significant differences between the EC and BDPN groups on day two of cultivation. On day three significant differences were found also between the EC and BDPN groups (p = 0.000). The embryonic developmental arrest was found only in PN embryos. CONCLUSION: The speed of the first cell cleavage is a useful additional criterion for the embryo selection for embryotransfer. EC embryos usually have better morphology and more blastomeres than the BPDN and PN ones.

Cleavage Stage, Ovum↗

PubMed1984

Cell relationships during aggregation between preimplantation embryos and teratocarcinoma-derived cells.

Cleavage-stage mouse embryos aggregate and form chimaeric blastocysts with embryonal carcinoma (EC) cells. We used scanning and transmission electron microscopy to study cell relationships during aggregate formation between 8-cell-stage embryos and F9 EC cells. Relations between heterotypic cells were similarly studied in aggregation experiments with embryos and teratocarcinoma-derived visceral (PSA5-E) and parietal (PYS-2) endoderm cells and in experiments with EC cells and endoderm cells. The embryos and F9 cells always adhered to each other and rapidly formed compacted aggregates. Numerous microvilli and cell processes, originating from both embryo and EC cells, extended between the two cell types during adhesion and early phases of aggregation. The aggregation process involved spreading of the blastomeres on the EC cells. Frequent adherent junctions and close contacts, including possible focal gap or tight junctions were observed between the embryo and F9 cells after 3 h of culture. Apparent gap or tight junctions were infrequent during the early phases of aggregation but during further culture, extensive typical gap junctions were also seen between embryo and EC cells. The embryos adhered only irregularly and loosely to PSA5-E and PYS-2 cells; this interaction never led to aggregate formation comparable to that seen in the experiments with embryos and EC cells. Close contacts but no gap or tight junctions could be observed between the embryo and endoderm cells. On the other hand, both PSA5-E and PYS-2 cells readily adhered to and aggregated with EC cells. The present results suggest that microvilli and cell processes mediate membrane interactions during adhesion and early phases of aggregation between embryos and EC cells. During aggregation, blastomeres spread over the EC cells, and rapid formation of adherent junctions and close contacts, including possible focal gap or tight junctions is involved during the early phases of this process. After this initial phase, typical gap junctions are also seen between the embryo and EC cells. Interestingly, adhesive properties of embryo and EC cells differ: the former aggregate only with EC cells, whereas the latter do so also with teratocarcinoma-derived visceral and parietal endoderm cells. Mechanisms operating in the morphogenetic movement of cells in this experimental setup may be involved also in the development of the blastocyst in vivo.

Animals↗

PubMed1995

Treatment with 5-aminolevulinic acid and photoactivating light causes destruction of preimplantation mouse embryos.

OBJECTIVE: To evaluate the direct effect of photodynamic treatment with 5-aminolevulinic acid (ALA) on preimplantation mouse embryos in an in vitro setting. DESIGN: Preimplantation mouse embryos were incubated with or without ALA for 5 hours and followed immediately by light exposure for 0, 5, or 15 minutes. Comparison of the viability and blastocyst formation was made among different treatment groups. SETTING: A conventional laboratory setting with embyro culture facilities. INTERVENTIONS: Female CD1 mice were superovulated with pregnant mare serum gonadotropin and hCG before mating. Four-and eight-cell embryos and compacted morulae were flushed from the oviducts and incubated with 0, 0.1, 0.5, 1.0, or 5.0 mM ALA for 5 hours. Embryos subsequently were exposed to photoactivating light for 0, 5, or 15 minutes. MAIN OUTCOME MEASURES: Microscopic assessment of embryos quality at 12 hours and determination of the percentage of embryos progressing to the blastocyst stage at 36 or 60 hours. RESULTS: Incubation of embryos with 0.1, 0.5, 1.0, 5.0 mM ALA without light resulted in 87.3% +/- 1.6%, 84.9% +/- 3.4%, 81.4% +/- 1.8%, and 82.8% +/- 4.7% of the embryos developing to blastocysts, respectively. In the absence of ALA, light exposure for 0, 5, or 15 minutes resulted in 93.8% +/- 2.3%, 92.3% +/- 2.2%, and 85.9% +/- 1.7% blastocyst formation. Combining treatment of ALA at the same concentrations with light resulted in 33.3% +/- 2.1%, 0.7% +/- 0.9%, 0%, 0% (5-minute light), 13.3% +/- 1.0%, 0%, 1.6% +/- 1.3%, 0% (15-minute light) blastocyst formation, respectively. When gross morphology was used to assess embryo viability at 12 hours, similar results were observed. Measurement of the fluorescent spectrum of embryos incubated with ALA indicated that protoporphyrin IX had been formed. CONCLUSION: Photodynamic ablation of mouse embryos was achieved with ALA under in vitro conditions. These results indicate that preimplantation mouse embryos are capable of converting ALA to the photosensitizer, protoporphyrin IX, and are susceptible to subsequent photoablation. A photodynamic effect on the embryo may be important to the successful application of this technique to the treatment of human ectopic pregnancy.

Aminolevulinic Acid↗

PubMed1986

Factors affecting survival and implantation of cryopreserved human embryos.

Five- to ten-cell embryos and expanded blastocysts from 68 patients were thawed in an attempt to establish pregnancy. Three fresh embryos had been replaced unsuccessfully in these patients. Forty-five patients had intact freeze-thawed embryos replaced and 12 became clinically pregnant. Ten of these pregnancies have now advanced beyond week 24. The preliminary results presented here demonstrate that significantly more expanded blastocysts survive cryopreservation than cleaving embryos. Faster-growing embryos did not survive better than slowly growing embryos, but the incidence of implantation was higher with faster-developing embryos. Significantly more blastocysts with a "normal" morphology survived cryostorage than those scored as "irregular" (77 versus 7%). A similar trend was observed when normal and irregular cleaving embryos were frozen, but the difference was not significant. The severity of contraction of blastocysts upon the addition of cryoprotectant increased the incidence of survival. The proportion of patients whose embryos had holes in the zona pellucida following cryopreservation was significantly higher when embryos were frozen at the cleaving stage (39%) rather than the expanded blastocyst stage (16%). Almost all embryos with damaged zonae degenerated. The proportion of cleaving embryos that survived cryostorage was inversely correlated with the number of follicles aspirated.

Blastocyst↗

PubMed2004

Number of good quality embryos on day 3 is predictive for both pregnancy and implantation rates in in vitro fertilization/intracytoplasmic sperm injection cycles.

OBJECTIVE: To evaluate the predictive value for implantation and pregnancy rates of the number of embryos that reach the eight-cell stage with less than 20% fragmentation (good quality embryos) on day 3. DESIGN: Prospective observational study. SETTING: Private IVF center. PATIENT(S): One hundred eighty-nine women undergoing IVF, or intracytoplasmic sperm injection with at least four zygotes, who have had three embryos transferred on day 3. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate and implantation rate. RESULT(S): All patients (n = 189) had 3 embryos transferred. The patients were divided in five groups according to the number of good quality embryos on day 3 (0, 1, 2, 3, >3; respectively). For the five groups, the pregnancy rates were 2.9%, 33.3%, 45.5%, 39.4%, and 64.3%; the implantation rates were 2.0%, 17.0%, 20.5%, 19.3%, and 35.7%. No statistically significant differences in pregnancy rate or implantation rate were found for groups 1, 2, and 3 (when 1, 2, or 3 good quality embryos were present). The data were also analyzed after pooling these three groups. A statistically significant difference in pregnancy rate and implantation rate was found for group 0 (no good quality embryos), groups 1, 2, and 3 (1, 2, or 3 good quality embryos), and group >3 (>3 good quality embryos). CONCLUSION(S): The number of good quality embryos available on day 3 is a strong predictive value for both pregnancy rate and implantation rate. When good quality embryos are present on day 3, only two embryos should be transferred to minimize multiple pregnancies.

Adult↗

PubMed2006

Early cleavage morphology affects the quality and implantation potential of day 3 embryos.

OBJECTIVE: To assess the development and implantation potential of early-cleaved embryos displaying various morphological patterns. DESIGN: Retrospective analysis. SETTING: Private IVF center. PATIENT(S): Embryos obtained from 1,556 transfer cycles were assessed. Early-cleaved embryos were grouped according to their cleavage patterns as: even (1,490); uneven (3,238); and fragmented (768), or according to nuclear morphologies as: mononucleation (2,008) and other nuclear morphologies; nonmononucleation (3,488). Seven thousand four hundred forty-five embryos were late cleaved. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryo quality, pregnancy (PR), and implantation rates. RESULT(S): Day 3 embryo quality was highest in evenly early-cleaved embryos and in those displaying mononucleation. Early-cleaved embryos displaying fragmentation and late-cleaved embryos yielded the poorest day 3 quality. Early cleavage cycles displayed higher PR and implantation rate than late cleavage with the exception of other nuclear morphologies, in which similar outcome was obtained. Mononucleated early-cleaved embryos implanted at a higher frequency than early-cleaved embryos displaying other nuclear morphologies. CONCLUSION(S): The morphology of early cleavage correlates to day 3 embryo quality and implantation rate.

Adult↗

PubMed2006

Deciding the fate of supernumerary frozen embryos: a survey of couples' decisions and the factors influencing their choice.

OBJECTIVE: To investigate the decisions that couples make regarding supernumerary frozen embryos, the factors influencing these decisions, and the degree of difficulty involved in reaching a decision; and to canvass attitudes toward donating embryos to stem-cell research. DESIGN: Anonymous postal survey. SETTING: A large, private IVF clinic in a major city in Victoria, Australia. PATIENT(S): A consecutive cohort of couples who contacted the Monash IVF clinic in relation to embryos in long-term storage. INTERVENTION(S): Subjects completed a survey regarding decisions about surplus frozen embryos. MAIN OUTCOME MEASURE(S): Couples' decision regarding supernumerary embryos and reasons for the decision, experience of deciding, and attitudes about embryo donation for stem-cell research. RESULT(S): Forty percent (123/311) returned completed questionnaires. The most common decision was donation to research (42%). Altruistic motives and desire not to waste embryos were determinants of embryo donation. Determinants of disposal were not wanting a full sibling to existing children and opposition of embryo research. Forty-five percent found deciding distressing. The majority (69%) approved of embryo donation to stem-cell research. CONCLUSION(S): Most couples preferred embryos to come to some use rather than being disposed of. Almost half the sample reported finding the decision making distressing. A majority approved of embryo donation for stem-cell research.

Adult↗

PubMed2007

[Embryo donation in France: practice and difficulties. Strasbourg's experience].

OBJECTIVE: The aim of this study was to present the situation of embryo donation in France and around the world, to expound the difficulties of its practice and the results obtained in our centre 3 years after the introduction of this procedure. PATIENTS AND METHODS: Embryo donation in France is controlled by implemented decrees published between 1999 and 2004. The couples, who have stored frozen embryos since at least two years, were contacted for a pluridisciplinary medical consultation. The indication of embryo donation was evaluated for the recipients through a pluridisciplinary approach. RESULTS: Among the interviewed couples, 16.7% have chosen embryo donation but only half of them have completed the procedure (6% of the couples with frozen embryos). The main indications for embryo donation were a double sterility, unexplained genetic disease, ART failures (poor fertilization or bad embryo quality) and oocyte donation when the delay was too long for the couples. The pregnancy rate was 28.6% after the 21 first embryo transfers. DISCUSSION AND CONCLUSION: The results of embryo donation confirm the international experience both considering the poor number of donated embryos, medical indications and results. Embryo donation has its place among ART techniques, but one should not ignore the general debate on ethical questions raised by this procedure.

Embryo Disposition↗

PubMed2005

Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen.

This scientific review was prompted by recent legislation to curtail the use of semen from potentially virus-infected bulls to produce embryos for import into the European Union. From studies in laboratory animals, humans and horses, it is apparent that viruses may sometimes attach to, or be integrated into, spermatozoa, although in domestic livestock, including cattle, this seems to be a rare phenomenon, and carriage of virus through the zona pellucida into the oocyte by fertilising sperm has never been described in these species. Four specific viruses; enzootic bovine leukosis (EBLV), bovine herpesvirus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV), all of which tend to cause subclinical infections in cattle, but which can occur in bovine semen, are examined with regard to the risks that use of infected semen might lead to production of infected embryos. With regard to in vivo-derived embryos, when internationally approved embryo processing protocols are used, the risks from EBLV- and BTV-infected semen are negligible, and the same is almost certainly true for semen infected with BoHV-1 if the embryos are also treated with trypsin. For BVDV, there is insufficient data on how the virus is carried in semen and how different BVDV strains can interact with sperm, oocytes and embryos. There is a potential, at least, that in vivo-derived embryos resulting from infected semen might carry BVDV, although field studies so far suggest that this is very unlikely. With regard to in vitro-produced embryos, use of semen infected with any of the four viruses, with the probable exception of EBLV, will often lead to contaminated embryos, and virus removal from these embryos is difficult even when the internationally approved embryo processing protocols are used. However, it has never been demonstrated that such embryos have resulted in transmission of infection to recipients or offspring.

Animals↗

PubMed2006

Quantification of embryo quality by respirometry.

It is generally accepted that assessment of embryo metabolism, in particular oxygen consumption, may improve embryo selection by identifying the embryos with higher developmental competence. Several methods have been employed to measure embryonic oxygen consumption, but most of them were detrimental to subsequent embryo development. Recently, we have introduced the Nanorespirometer system, which is a non-invasive and highly sensitive technology developed for the individual measurement of embryonic respiration rates. This technology is able to perform single measurements at a fixed time or stage of embryonic development without adversely influencing embryo viability. Concomitantly, and based on the same principles, a second technology -- the Embryo Respirometer -- has been developed. The Embryo Respirometer allows the continuous measurement of individual respiration rates with simultaneous acquisition of digital images of each embryo, during the entire culture period (6-7 days). In this review, both technologies are described and their potential use as diagnostic tools for improving embryo selection in bovine and human following IVF treatments is discussed. Correlations between respiration rates of individual embryos and other parameters such as morphological quality, sex, stage of development, kinetics, diameter, expression of key metabolic genes and subsequent viability following embryo transfer are also examined. On the basis of the results obtained, it is postulated that assessment of embryonic respiration rates in association with other viability parameters allows for a more accurate embryo evaluation, both under clinical and research conditions.

Animals↗

PubMed2003

A meta-analysis of ultrasound-guided versus clinical touch embryo transfer.

OBJECTIVE: To determine the relative efficacy of ultrasound-guided embryo transfer and embryo transfer by clinical touch alone. DESIGN: Systematic review and meta-analysis of randomized, controlled trials comparing ultrasound-guided embryo transfer with embryo transfer by clinical touch alone. SETTING: Infertility centers providing treatment with in vitro fertilization/embryo transfer. PATIENT(S): Women undergoing embryo transfer. INTERVENTION(S): Embryo transfer with or without transabdominal ultrasound guidance. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate and embryo implantation rate. RESULT(S): A total of eight prospective controlled trials were identified. Of these studies, four were nonrandomized or quasi-randomized and four were genuinely randomized. Meta-analysis demonstrated a significantly increased chance of clinical pregnancy following ultrasound-guided embryo transfer in all studies and in the genuinely randomized subgroup. The embryo implantation rate was also significantly increased following ultrasound-guided embryo transfer. CONCLUSION(S): Ultrasound-guided embryo transfer significantly increases the chance of clinical pregnancy and significantly increases the embryo implantation rate.

Controlled Clinical Trials as Topic↗

PubMed1995

Ethical and legal issues in human embryo donation.

OBJECTIVE: To identify main ethical and legal issues that arise with donation of embryos left over from IVF treatments of infertility or created from separate gamete donations. DESIGN: Analysis of ethical commentary, advisory committee reports, statutes, court cases, and legal commentary relating to gamete and embryo donation and assisted reproduction to assess their effect on donation of created or leftover embryos. RESULTS: Donation of surplus embryos or embryos created from separate gamete donations would help a subset of infertile couples to form families. A program undertaking embryo donation will have to coordinate the donation of embryos from its own patients or other programs or arrange for separate gamete donations to form embryos. The main ethical issues concern the effect on offspring, consent and counseling of donors and recipients, avoidance of mixing embryos or gametes from different sources, and payment of donor expenses. The main legal issues concern whether embryo donation is viewed as gamete donation or adoption; the rearing rights and duties of donors and recipients in resulting offspring; liability; and compensation issues; and the legality of monetary compensation for donors. Donation of embryos for research raises separate issues. CONCLUSION: Human embryo donation is an ethically and legally acceptable way for infertile couples to form families.

Commodification↗

PubMed2003

Vitrification of rat embryos at various developmental stages.

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.

Animals↗

PubMed2001

Reduction of infectious epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos by non-specific protease.

Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced bovine embryos than to zonae of in vivo derived embryos. Currently, the International Embryo Transfer Society recommends that all in vivo derived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contrast to in vivo derived embryos, this method is not effective for disinfecting in vitro produced embryos. Our hypothesis was that a more potent, non-specific protease from Streptomyces griseus (S. griseus) would provide a more effective treatment for virus removal from in vitro produced bovine embryos. Bovine oocytes were matured, fertilized, and cultured in completely defined in vitro conditions. Zygotes were washed according to the procedure outlined by the International Embryo Transfer Society, replacing trypsin with the experimental protease. Experimental incubations were with 0.1% (4 units/ml) protease for 0, 30, 45, 60 and 75s intervals. Embryos were able to withstand exposure to this enzymatic treatment for only 45s before their developmental potential was significantly reduced; 60s exposure was detrimental (P<0.05). Oocytes were exposed to epizootic hemorrhagic disease virus serotype 2 (EHDV-2, 10(6) TCID(50)/ml) during in vitro maturation. Resulting zygotes were washed according to the International Embryo Transfer Society procedure and either exposed to trypsin or protease. Exposure to EHDV-2 prevented cumulus expansion and markedly reduced embryonic development (P<0.05). There were no differences in development among virus exposed groups receiving no treatment or treatment with trypsin or protease. However, proportions of infected embryos were reduced after protease treatment versus positive controls and trypsin treated embryos.

Animals↗

PubMed2003

The current status and future of commercial embryo transfer in cattle.

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen currently is available in only a few countries and on an extremely limited basis. As of yet, all programs involving the production of transgenic cattle are experimental in nature.

Animal Rights↗