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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.

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 versus outpatient, day post-ovulation), embryo (interval from ICSI to blastocyst formation) and management factors (season when Ovum Pick-Up 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. This article is protected by copyright. All rights reserved.

The cloning of equines by somatic cell nuclear trans- fer (SCNT) was first reported in 2003 (Galli et al., 2003; Woods et al., 2003), 6 years after the birth of Dolly the sheep (Wilmut et al., 1997). Amongst the mammalian spe- cies that have been cloned (Campbell et al., 2007), equines are among the last. In general, the application of assisted reproduction technologies in equines has lagged behind that of other livestock species (Galli et al., 2007) in the poor interest expressed by the industry (still today, for example, the thoroughbred does not allow artificial insemi- nation) and also the limited resources devoted to this kind of research and the few laboratories working in this area. In fact the first equine clone, a mule (Woods et al., 2003), was obtained from in vivo matured oocytes that were transferred back to the oviduct of a synchronized recipi- ent mare immediately after nuclear transfer and activation. This approach was successful because it limited to a mini- mum the time exposure to the in vitro environment. This approach of using in vivo matured oocytes was, however, not sustainable from a practical point of view. In contrast, the first cloned horses (Galli et al., 2003; Lagutina et al., 2005) and those that followed later (Hinrichs et al., 2006, 2007) were all derived from abattoir-recovered and in vitro matured oocytes that, following enucleation and embryo reconstruction, were cultured in vitro to the blastocyst stage prior to non-surgical embryo transfer to a recipient mare. This method could be applied only after the develop- ment of the procedures for in vitro maturation of oocytes, fertilization by ICSI, and in vitro culture of embryos capable of establishing normal pregnancies and generat- ing offspring (Galli et al., 2007; Hinrichs, 2010). In addi- tion to the refinement of the in vitro protocols for oocyte maturation and embryo culture, SCNT protocols had to be adapted and optimized for the equine, which, because of its anatomical and physiological features, provides a lim- ited number of oocytes per ovary when compared to cat- tle or pigs. The protocols currently applied for nuclear transfer are based on a zona-free system (Lagutina et al., 2007) to enhance cell fusion or a piezo-electric device (Westhusin et al., 2003) for enucleation and microinjec- tion of the nucleus directly into the oocyte. Reconstructed embryos also require a specific activation protocol (Hinrichs et al., 2006; Lazzari et al., 2002).

Equine Assisted Reproduction and Embryo Technologies

Clinical application of Ovum Pick Up, Intracytoplasmic sperm injection and Embryo culture in equine reproduction

The use of 3I medium for the derivation of bovine embryonic stem cells

Differentiation of early neuroectodermal precursors derived from human embryonic stem cells into neural crest cells and dopaminergic neurone

Offspring of pre-determined sex has been obtained in several species with the use of sex-sorted semen by flowcytometry followed by artificial insemination or assisted reproduction techniques. Nevertheless in some species such as pig, sheep and horse efficiency is highly reduced compared to conventional AI due to the high number of sperm required for insemination. To obtain offspring from semen available in very limited amounts and with poor motility, as always occurs with sex-sorted frozen stallion semen, the application of the in vitro assisted reproductive techniques, in particular intracytoplasmic sperm injection has become a real option. The scope of this work was to investigate the use of sex-sorted frozen stallion semen for the production of embryos in vitro following ICSI, in vitro culture, freezing and thawing, and their viability after transfer to recipients to produce offsprings. Ejaculates were collected from 2 Standardbred stallions of proven fertility (A, B). Only the ejaculates containing >60% motile spermatozoa after dilution were used for subsequent assessment. Briefly semen samples were diluted, stained with Hoechst 33342 and food dye and sorted with a MoFlo SX® flow cytometer/sperm sorter. Flow-cytometrically sorted spermatozoa were deflected into polypropylene tubes containing 500 μl of 2 % Tes-Tris-egg yolk buffer, than the collected semen was centrifuged, diluted in freezing medium and frozen. Sex-sorted and control non-sexed frozen semen (two stallions of in vitro proven fertility, C, D) was thawed, centrifuged on a Percoll gradient, washed and diluted 1:1 in PVP before ICSI. Oocytes were collected from ovaries of slaughtered mares at the beginning of the breeding season and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen in glycerol. Six embryos were subsequently thawed and non-surgically transferred in naturally cycling synchronous recipient 5 days after spontaneous ovulation. Overall 70, 58, 30 and 15 oocytes were injected with sex-sorted and control frozen semen from stallion A, B, C, D respectively. Mean cleavage rates were 20.31% for sorted sexed semen and 71.11% for control showing a significantly lower cleavage rate for sexed semen. This difference was reflected on the number of blastocyst obtained (4.18% vs 20.00%). Out of 6 frozen-thawed embryos transferred we obtained 4 pregnancies, one was lost at 21 days, a second was pharmacologically aborted at 21 days and two were maintained to go to term (one male and one female are currently in the 8th month of gestation). In conclusion, in this study we demonstrated that sex-sorted sperm can be used for producing blastocysts able to establish pregnancies at high rate when transplanted in recipient mares. However the efficiency of the system is low due to the very low cleavage rate obtained with such sex-sorted frozen thawed semen.

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.

We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.