Identified and unidentified challenges for reproductive biotechnologies regarding infectious diseases in animal and public health.

The aim of the present paper is to review the known and theoretical risks for in vivo derived and in vitro produced embryos as well as for nuclear transferred or transgenic embryos in terms of animal diseases or diseases of public health consequence. For in vivo derived embryos, a considerable number of experiments and scientific investigations have resulted in recommended guidelines and procedures that ensure a high level of safety. The effectiveness of these measures has been validated by field experience with the safe transfer of several million embryos over the past three decades. In vitro produced embryos have several characteristics that differentiate them from the former, in particular a structure of the zona pellucida that results in a more frequent possible association of pathogens with the embryo. However, the guidelines prescribed by the IETS, the international standard setting body (OIE) and existing national regulatory frameworks are in place to minimize the risk of disease transmission. No specific public health risks have been identified to date with respect to in vivo or in vitro derived embryos. In regard to nuclear transferred and transgenic embryos, theoretical risks have been identified in relation to the potential effects on some intrinsic viruses such as endogenous retroviruses but very little targeted experimental work has been carried out on infectious diseases that could have adverse consequences on animal or human health. Although there has been no report of such adverse consequences associated with the limited number of animals produced to date by such reproductive technologies, a precautionary approach is warranted given the potential negative impacts and it would be prudent to restrict at this stage, the international movement of such "manipulated" embryos.     

In vitro technologies related to pig embryo transfer

Embryo transfer in swine (ETS) has been used for commercial and breeding application only to a limited extent. However this technique is an essential prerequisite for the application of new reproductive techniques in pigs. This paper will give an overview on steps of pig embryo transfer including selection and stimulation of donor sows, recovery of embryos, embryo handling and the transfer of recovered embryos into recipients. Furthermore the current status and further application of ET related in vitro technologies in pig production are described.     

Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study

The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I) blastocysts were compared for the occurrence of apoptosis in blastomeres. The total number of cells per embryo and the number of cells with damaged plasma membranes, fragmented DNA and fragmented nuclei per embryo were assessed in selected blastocysts by combining Ethidium homodimer (EthD-1), terminal dUTP nick end labeling (TUNEL) and Hoechst 33342 staining. In general, the level of blastomere apoptosis was low. A higher level of apoptosis was observed in in vitro produced equine, porcine and bovine blastocysts compared to their in vivo counterparts. Interestingly, 4 of the initially selected 29 bovine in vitro produced blastocysts exhibited extensive signs of apoptosis affecting the inner cell mass (ICM), which is not compatible with a viable conceptus. Repeated occurrence of this observation may explain the lower ET outcome of in vitro produced bovine embryos compared to in vivo produced embryos. It is concluded that, although in morphologically high quality blastocysts of several farm animal species a significant difference exists in the percentages of apoptotic cells between in vivo and in vitro produced embryos, the incidence of apoptosis at the blastocyst stage is at such a low level that it cannot reflect the substantial differences in embryo viability that have been described between in vivo and in vitro produced blastocysts following ET.     

Infectious agents in bovine embryo production: hazards and solutions

Infectious agents in systems for producing bovine embryos might reduce the number and quality of embryos generated, result in transmission of disease to recipients and offspring, or confound findings of research. Embryo-associated pathogens might also jeopardize human health when the goal of embryo production is creating transgenic animals intended to be a source of pharmaceuticals or organs. This paper addresses risks and resulting hazards of pathogen and microbial contaminant introduction into in vivo or in vitro embryo production systems. Additionally, methods for prevention and quality control are discussed.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Improving fertility in beef cow recipients

In the 1970s, bovine embryo transfer (ET) shifted from research in a laboratory environment to commercialization of this technology for beef producers. With the quarantine requirements and expense of importing Continental breeds of cattle from Europe, embryo transfer became the logical means to reproduce greater numbers of these animals at a lower cost. The ET industry grew very rapidly and soon would become what it is today, a common practice utilized by select ranchers and breeders. Research over the years has primarily focused on methods to increase the number of ovulations and fertilized ova from the donor female, but the total number of transferable embryos has not changed markedly in the last 20 years. More recent advances have been in the area of in vitro production of embryos that allow for greater numbers of embryos to be produced and easier accessibility to incorporate technologies such as sexed sperm, sperm injection, or transgenics. This paper will focus on the second part of the equation, the recipient, and decisions that will enable both the customers and practitioners to most efficiently utilize embryos from superovulation, in vitro production, or nuclear transfer, so that the maximum number of pregnancies can be produced.     

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.     

In vitro embryo production in buffalo species: state of the art.

In the last several years, there has been an increasing interest in in vitro embryo production (IVEP) technologies for faster propagation of superior germplasm in buffalo because of the low efficiency of superovulation (SO) and embryo transfer (ET) programs in this species. Although the IVEP efficiency has improved, embryo yield and development to term are still very low. This paper reviews the progress made in optimizing the IVM, IVF, and IVC systems. It also highlights the importance of embryo cryopreservation, which might critically contribute to the diffusion of ET procedures in the field. The acquisition of more information on embryo physiology, metabolism, and culture requirements in this species is critical to optimize the efficiency of advanced reproductive strategies. Further studies are also needed to improve the cryopreservation of IVEP embryos. The second part of the work underlines the potential impact of ovum pick-up (OPU) technique combined with IVEP on genetic improvement of buffalos. The OPU technique is a non-invasive and repeatable procedure for recovering large numbers of meiotically competent oocytes from antral follicles of live animals. Our experience, in buffalo, has demonstrated that OPU is superior to SO because it can yield more transferable embryos (TE) per donor on a monthly basis (2 TE vs 0.6, respectively). Therefore this technology has great potential to improve the genetic progress of buffalo through the maternal lineage.     

Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility. A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare. The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitro and in vivo screening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.     

Risk assessment of transmission of bovine viral diarrhea virus (BVDV) in abattoir-derived in vitro produced embryos

Bovine virus diarrhea virus (BVDV) is a pathogen of the bovine reproductive system causing reduced conception rates, abortions and persistently infected calves. Most if not all strains of BVDV are transmissible by natural mating and AI. For international trade, it is recommended that in vitro fertilized embryos be washed according to the IETS Manual. However, BVDV may not be entirely washed out, resulting in possible transmission risks to recipients. Donor cows, donor bulls and biological agents are all possible sources of contamination. The process for producing in vitro produced (IVP) embryos is complex and non-standard, and some procedures can contribute to spread of BVDV to uninfected embryos. The structure of the zone pellucida (ZP) of IVP embryos permits adherence of BVDV to the ZP. To estimate the risk of producing infected recipients and persistently infected calves from abattoir-derived IVP embryos, a quantitative risk assessment model using Microsoft Excel and Palisade @Risk was developed. Assumptions simplified some of the complexities of the IVP process. Uncertainties due to incomplete or variable data were addressed by incorporating probability distributions in the model. Model variables included: disease prevalence; the number of donor cows slaughtered for ovaries; the number of oocytes collected, selected and cultured; the BVDV status of ovaries, semen, biological compounds and its behavior in the IVP embryo process. The model used the Monte Carlo method to simulate the IVP process. When co-culture cells derived from donor cows of unknown health status were used for in vitro culture (IVC), the probability of a recipient cow at risk of infection to BVDV per oocyte selected for IVP processing averaged 0.0006. However, when co-culture free from BVDV was used, the probability was 1.2 x 10(-5). Thus, for safe international trade in bovine IVP embryos (i.e. negligible risks of transmission of BVDV), co-culture cells, if used during IVC for producing IVP embryos, should be disease-free.     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

广西巴马小香猪体细胞克隆

本研究旨在检验新生广西巴马小香猪肾脏成纤维细胞支持克隆胚胎完全的体内发育潜能,亦即能通过其构建出存活的克隆猪,从而为克隆技术在广西巴马小香猪资源保存和开发上的应用奠定基础。首先制备新生雄性广西巴马小香猪肾脏成纤维细胞,用其制备体细胞核移植胚胎,追踪观察体细胞核移植胚胎体外发育潜能,最后通过胚胎移植检验其完全的体内发育潜能。实验结果表明,制备的新生雄性广西巴马小香猪肾脏成纤维细胞具有良好的细胞增殖活性,用其制备的体细胞核移植胚胎分裂率和囊胚率分别为77.7%(334/430)和16.5%(71/430),将1 658枚克隆胚胎移植给6头代孕母猪,其中2头妊娠并最终产下8头存活雄性克隆小猪和3头死胎,整体克隆效率为0.66%,存活克隆猪健康状况良好。本研究表明,新生猪肾脏成纤维细胞是一种理想的用于生产体细胞克隆广西巴马小香猪的细胞资源。     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.     

Nuclear transfer and electrofusion in bovine in vitro-matured/in vitro-fertilized embryos: Effect of media and electrical fusion parameters

In this study, micromanipulation and electrofusion conditions for the cloning of in vitro-produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 μsec. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle. © 1993 Wiley-Liss, Inc.     

Factors affecting success of embryo collection and transfer in large dairy herds

Our objective was to evaluate factors that affected the success of embryo transfer programs in large dairy herds. Non-lactating donor cows produced a larger number of ova/embryos (P<0.01) and viable embryos (P<0.01) than lactating cows. The interaction between season and donor class was correlated with the proportion of ova/embryos classified as fertilized (P=0.03), because lactating donors had fewer fertilized ova in the summer. There was no correlation between 305-day mature equivalent milk yield and response to superstimulation. Although the interval between superstimulation protocols was correlated with the number of ova/embryos (P=0.03), there was no correlation with the number of viable embryos. Pregnancy per embryo transfer (P/ET) in heifer recipients was correlated with embryo quality grade (P<0.01), season (P=0.04), and whether embryos were fresh or frozen/thawed (P<0.01). Lactating recipient cows tended to have a lower rate of P/ET during the summer (P=0.12 to P=0.08). Synchronization protocols tended to be (P=0.06; Herd 1) or were (P=0.02; Herd 2) correlated with P/ET. Lactating cows receiving vitrified IVF embryos had a lower (P=0.01) P/ET than those receiving fresh IVF embryos, especially in the summer (P=0.09). Milk yield was not correlated with P/ET. The use of heat abatement systems is critical to improve embryo production and P/ET. Synchronization protocols that optimized synchrony of ovulation may increase fertility of recipient cows and eliminate the need for estrous detection.     

Nuclear transfer of mouse follicular epithelial cells pretreated with spermine, protamine, or putrescine

The in vitro and in vivo developmental potential of nuclear transferred embryos receiving follicular epithelial cells pretreated with spermine (5 and 20 mM), protamine (0.25 and 25 mg/ml), or putrescine (1 and 100 microg/ml) at room and reduced temperatures was examined in the mouse. The pretreated donor cells were first fused with enucleated oocytes, and then nuclei from reconstituted eggs at the two-cell stage were fused with the enucleated fertilized two-cell embryos. The proportion of reconstituted embryos that developed into blastocysts was not significantly different among groups. After transfer to recipients, implantation rates were not different between groups and fetuses were obtained in protamine- and spermine-treated groups as well as in control groups. These results demonstrate that pretreatment of nuclear donor cells with spermine, protamine, or putrescine does not enhance the developmental potential in vitro or in vivo in the mouse. J. Exp. Zool. 289:208-212, 2001.     

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.     

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.