Speed of in vitro embryo development affects the likelihood of foaling and the foal sex ratio.

The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient day after ovulation at transfer. The odds ratio for foaling was ~0.63 for transfer of Day 8 (46%) compared with Day 7 (56%) IVP blastocysts. The highest likelihood of pregnancy (72%) and foaling (60%) was observed when IVP blastocysts were transferred to recipient mares on Day 4 after ovulation. Finally, the sex (colt:filly) ratio was higher after transfer of Day 7 (71%:29%) than Day 8 (54%:46%) IVP blastocysts, suggesting that the speed of embryo development is sex dependent. In conclusion, the speed of invitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.

Modeling amyotrophic lateral sclerosis in hSOD1 transgenic swine.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS.

Somatic cell nuclear transfer and transgenesis in large animals: current and future insights.

Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications.

Embryonic genotype and inbreeding affect preimplantation development in cattle.

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes.

Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were labelled for connexin-43 or injected with Lucifer yellow dye to determine whether the integrity of the gap junctions between an oocyte and its cumulus was compromised by vitrification. Finally, the effect of denuding immature and mature oocytes on their ability to withstand vitrification was examined. Cryopreserving immature COCs increased the number of dead cumulus cells (13 vs 2.6% for controls; P<0.05). However, an increased proportion of dead cumulus cells did not affect post-warming maturation rates (approximately 30% MII) presumably because dead cells were located at the periphery of the cumulus mass and cumulus-oocyte gap junction communication was not disrupted. Moreover, cumulus removal prior to IVM or vitrification indicated that while the cumulus does protect immature oocytes during vitrification it does so by mechanisms other than support during maturation. Cumulus presence was also found to protect mature equine oocytes against vitrification-induced damage since cumulus-enclosed MII oocytes preserved their meiotic spindle quality better during vitrification than denuded oocytes (38.1 vs 3.1% normal spindles; P<0.05).

Factors affecting the likelihood of pregnancy and embryonic loss after transfer of cryopreserved in vitro produced equine embryos.

BACKGROUND: In vitro embryo production (IVEP) is increasingly popular but data assessing the outcome of transferred embryos are scarce. OBJECTIVES: To determine the likelihood of pregnancy and embryonic loss after transfer of frozen-thawed IVP embryos and identify factors influencing success. STUDY DESIGN: Retrospective clinical study. METHODS: Blastocysts (n = 261) were produced from immature oocytes of Warmblood mares (n = 116) by Intracytoplasmic Sperm Injection (ICSI) and in vitro culture, and cryopreserved. Thawed IVP embryos were transferred into recipient mares on day 4, 5 or 6 after ovulation. The influence of donor mare (age, reproductive history), recipient mare (age, reproductive status, management; in-house vs. outpatient, day post-ovulation), embryo (interval from ICSI to blastocyst formation) and management factors (season when ovum pickup was performed, year and method of transfer) on likelihood of pregnancy and embryonic loss was examined, and the developmental stage of the IVP embryo at the time of transfer was estimated. RESULTS: The percentage of mares pregnant 7-10, 23 and 37 days after transfer was 56% (147/261), 49% (129/261), and 48% (124/261), respectively. Development of IVP embryos after transfer equated to day 5 or 6 in vivo embryos. With the exception of year of transfer, none of the factors had an impact on the likelihood of pregnancy or embryonic loss. Nevertheless, the likelihood of pregnancy tended to be lower for IVP embryos from infertile mares or when embryos were transferred into recipient mares on day 6 after ovulation rather than on day 4 or 5. Finally, the diameter of the embryonic vesicle 7 days post transfer was lower for pregnancies that were lost compared to those that were maintained. MAIN LIMITATIONS: Small sample size in some of the donor and recipient mare categories. CONCLUSIONS: Cryopreserved IVP embryos should be transferred into recipient mares on day 4 or 5 after ovulation and a slower rate of post transfer vesicle expansion indicates a higher risk of subsequent embryonic loss The Summary is available in Portuguese - see Supporting Information.

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

QSAR study for a novel series of ortho disubstituted phenoxy analogues of alpha1-adrenoceptor antagonist WB4101.

On the basis of the affinities at the alpha1a-, alpha1b- and alpha1d-adrenoceptors and the 5-HT1A receptor of a previous series of sixteen 2-[(2-phenoxyethyl)aminomethyl]-1,4-benzodioxanes ortho monosubstituted at the phenoxy moiety, a number of ortho disubstituted analogues were designed, synthesized in both the enantiomeric forms and tested in binding assays on the same receptors. The affinity values of the new compounds 1-11 were compared with those of the enantiomers of the 2,6-dimethoxyphenoxy analogue, the well-known alpha1 antagonist WB4101, and of the ortho monosubstituted derivatives, suggesting some distinctive aspects of the interaction of the phenoxy moiety, in particular with the alpha1a-AR and the 5-HT1A receptor, of the monosubstituted and the disubstituted compounds. A classical quantitative structure-activity relationship (Hansch) analysis was applied to the whole set of the S enantiomers of the ortho mono- and disubstituted WB4101 analogues (26 compounds), finding a very good correlation for the alpha1a affinity. For this latter, a significant parabolic relationship was also found with the volume of the two ortho substituents. Diametrically opposite, the same relationships for the 5-HT1A exhibit low or insignificant correlation coefficients.

Differential Response of Human Embryonic Stem and Somatic Cells to Non-Cytotoxic Hydrogen Peroxide Exposure: An Attempt to Model In Vitro the Effects of Oxidative Stress on the Early Embryo.

Human Embryonic Stem Cells (hESCs) potentially offer a unique in vitro model to study how an adverse environment during the early developmental stages post-fertilization can affect the physiology of the undifferentiated embryonic stem cells existing in the early embryo and predispose to long term effects on the offspring, according to the Developmental Origins of Health and Disease (DOHaD) concept. A number of unfavourable conditions can affect the development of the early embryo inducing oxidative stress both in vivo, for instance in gestational diabetes and in vitro, when embryos are derived from Assisted Reproductive Technologies (ART). Therefore, the aim of this study was the development of a novel in vitro model to analyse the effects of oxidative stress and the antioxidant response against Reactive Oxygen Species (ROS) in embryonic stem cells in comparison with somatic cells, fibroblasts and endothelial cells. To this purpose we designed an in vitro protocol based on hydrogen peroxide (H2O2) treatment of 72 h, in order to better resemble the period of embryonic development from the early cleavages to the blastocyst stage. We demonstrate that H2O2 treatment induces the modification of crucial oxidative stress biomarkers like ROS and lipid peroxidation levels, and mobilizes several antioxidant enzymes through NFkß translocation. Moreover we show differences between somatic and embryonic cells in their antioxidant response towards H2O2 induced damage. Therefore this study presents a promising in vitro model to investigate the effects of oxidative stress conditions on early human embryonic cells.

Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.

Susceptibility of bovine mesenchymal stem cells to bovine herpesvirus 4.

Bovine herpesvirus 4 (BoHV-4) is a gamma herpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbour persistent BoHV-4. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of adjacent haematopoietic precursors, such as macrophages, the interaction between BoHV-4 and mesenchymal stem cells was investigated. Primary bovine mesenchymal stem cells were highly permissive to support full replication of BoHV-4. This finding could be considered a new important step in studies on the potential pathogenesis related to BoHV-4.

Bovine embryo technologies.

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Production and quality of bovine oocytes and embryos.

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.

Isolation and in vitro characterization of bovine amniotic fluid derived stem cells at different trimesters of pregnancy.

Amniotic fluid (AF) is a source of multipotent mesenchymal stem cells (MSCs), very promising cells for tissue engineering in clinical application. The aim of this work was to isolate and characterize cells isolated from bovine AF as alternative sources of primitive multipotent stem cells in a species that could be a large-animal model for biomedical and biotechnology researches. Samples were recovered, at slaughterhouse, from 39 pregnant cows at different trimesters of pregnancy and cells were cultured in vitro. At passages (P) 3 and 7 differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced. Flow cytometry analysis for CD90, CD105, CD73, CD44, CD34, CD45 and CD14 was performed, immunocytochemistry (ICC) for Oct4, SSEA4, α-SMA, Vimentin, N- and E- Cadherin and CK and qPCR analysis for OCT4, NANOG and SOX2 were carried out. The cell yield was significantly higher in the first trimester compared to the second and the third one (P < 0.05). Cells were isolated from 25/39 samples and cell population appeared heterogeneous. Two main cell types were identified in samples from all trimesters: round- (RS) and spindle-shaped (SS) cells. 17/25 samples showed both populations (mixed, MX). Both cell types showed MSC-markers and differentiation capability with some variability related to the passages. The SS-population also expressed low levels of stemness markers such as NANOG and SSEA4 but not OCT4. Bovine AF shows a heterogeneous cell population containing also MSCs, multipotent cells that represent an intermediate stage between embryonic stem cells and adult ones.

Characterisation of a neural teratogenicity assay based on human ESCs differentiation following exposure to valproic acid.

The development of in vitro testing strategies for chemical and drug screening is a priority need in order to protect human health, to increase safety, to reduce the number of animals required for conventional testing methods and finally to meet the deadlines of current legislations. The aim of this work was to design an alternative testing method based on human embryonic stem cells for the detection of prenatal neural toxicity. For this purpose we have created a model based on the generation of neural rosettes, reproducing in vitro the gastrulation events recapitulating the formation of the neural tube in vivo. To validate the model we have exposed this complex cell system to increasing concentrations of valproic acid, a known teratogenic agent, to analyse the morphological and molecular changes induced by the toxicant. Specific assays were applied to discriminate between cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with a microarray Affimetrix platform and validated by quantitative real time RT-PCR for the expression of genes involved in early neural development, neural tube formation and neural cells migration, key biological processes in which the effect of valproic acid is most relevant. The results demonstrated that neural rosette cells respond to valproic acid exposure with molecular and morphological changes similar to those observed in vivo, indicating that this method represents a promising alternative test for the detection of human prenatal neural toxicity.

Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species.

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes.

Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified-warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.

Cumulus-oocyte communications in the horse: role of the breeding season and of the maturation medium.

Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non-breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non-breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non-breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the non-breeding season. During the non-breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non-breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely.