Morphological characteristics of mule conceptuses during early developmentîen

Hybrids between species are often infertile and extremely rare among mammals. Mules, i.e. crossing between the horse and the donkey, on the other hand are very common in agricultural and leisure practices due to their enhanced post-natal physical characteristics that is believed to occur for outbreeding or hybrid vigor. Since no reports are availableon the effects of hybrid vigor during early development, this study focused on characterizing the intrauterine development of mule conceptuses during critical embryo-to-fetus transition period. Nine embryos and fetuses of early gestation, obtained after artificial insemination and transcervical flushing, were evaluated by means of gross anatomy and histology and compared to data available for the equine. We found that some events, such as C-shape turning, apearence of branchial archs, limb and tail buds, formation of primary and secondary brain vesicles, heart compartmentalization, and development of somites, occurred slightly earlier in the mule. Nonetheless, no major differences were observed in other developmental features, suggesting similarities between the mule and the horse development. In conclusion, these data suggest that the effect of hybrid vigor is present during intrauterine development in the mule, at least with regard to its maternal parent.(AU)

Morphological characteristics of mule conceptuses during early developmentîen

Hybrids between species are often infertile and extremely rare among mammals. Mules, i.e. crossing between the horse and the donkey, on the other hand are very common in agricultural and leisure practices due to their enhanced post-natal physical characteristics that is believed to occur for outbreeding or hybrid vigor. Since no reports are availableon the effects of hybrid vigor during early development, this study focused on characterizing the intrauterine development of mule conceptuses during critical embryo-to-fetus transition period. Nine embryos and fetuses of early gestation, obtained after artificial insemination and transcervical flushing, were evaluated by means of gross anatomy and histology and compared to data available for the equine. We found that some events, such as C-shape turning, apearence of branchial archs, limb and tail buds, formation of primary and secondary brain vesicles, heart compartmentalization, and development of somites, occurred slightly earlier in the mule. Nonetheless, no major differences were observed in other developmental features, suggesting similarities between the mule and the horse development. In conclusion, these data suggest that the effect of hybrid vigor is present during intrauterine development in the mule, at least with regard to its maternal parent.

Derivation and potential applications of pluripotent stem cells for regenerative medicine in horses

Background: The ability to create tissues using pluripotent stem cells to repair or replace tissue lost due to damage, i.e. regenerative medicine, is developing very rapidly in many fields of human medicine. For veterinarians, regenerative medicine has focused mainly in the use of stem cells for arthritis and tendon ligament repair, indicating a need for treating musculoskeletal injuries. Our objective is to review the available approaches being used to derive pluripotent stem cells and discuss their potential use for regenerative medicine in the horse. Review: Adult adipose- and bone marrow-derived mesenchymal stem cells (MSC) are being used in practice to treat injuries in horses. However, there is scarce scientific evidence of their effectiveness and little is known of the mechanisms by which such cell preparations improve the healing process. For instance, although early healing response of articular cartilage injury was improved by treatment with injection of MSC, they did not enhance the long-term tissue response, indicating that cell proliferation was attenuated. Better protocols for the isolation and clinical testing of equine MSC are required to confirm healing properties. In contrast to MSC, embryonic stem cells (ESC) derived from the inner-cell- mass (ICM) of blastocyst stage embryos carry the ability to proliferate indefinitely in vitro and, given appropriate and favorable conditions, can differentiate into any tissue in the body. Parthenogenesis (PG) and somatic cell nuclear transfer (SCNT) are used to obtain a genetic match to the host animal and, thereby, eliminate the risk of inducing immune rejection of the grafted tissue. However, apart from the typical markers of pluripotency, equine ESC also express markers of trophoblastic tissues, indicating that they are different and possibly less able to differentiate than the ESC lines obtained in other species. Consequently, further studies are underway to identify conditions to obtain fully pluripotent ESC lines from equine SCNT embryos. To overcome the limitations of ESC lines derived from equine embryos, induced pluripotent stem cells (iPSC) were derived using a piggyBac transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally controlled fashion. Our established fetal-derived iPSC lines express hallmark pluripotency markers, display a stable karyotype after prolonged culture, and are able to form teratomas in immunodeficient mice containing tissues from all three embryonic layers. By establishing a protocol for deriving stable iPSC lines in the horse, we expect that new opportunities will be shortly developed for regenerative therapies in this species. Conclusion: It is possible to derive autologous pluripotent stem cells in horses by using both ESC and iPSC-derived approaches. Although ESC lines are generally the gold standard of pluripotency, further research is required to improve the proliferative and pluripotency characteristics for clinical applications. On the other hand, equine iPSC show excellent stability during prolonged in vitro culture and have the capacity to differentiate into the three germ layers in vivo, suggesting that they could soon be used in pre-clinical trials. Therefore, further studies need to be performed to establish reliable protocols for assessing the regenerative properties of iPS and ESC for equine muscle-skeletal injuries.

Proteomics, metabolomics and lipidomics in reproductive biotechnologies: the MS solutions

Background : A broader view of living systems complexity is bringing important contributions to biological sciences, since the genome expression is affected by other classes of molecules, which in their turn interact themselves in cellular metabolic pathways and biochemical networks. This level of information has been made possible by the emergence of the omic strategies, such as proteomics, metabolomics and lipidomics, that are mainly based on mass spectrometry (MS) platforms. MS has presented an incredible development over the last years, evolving to a powerful and universal analytical technique. Its ability to analyze proteins and small molecules such as lipids, sugars and metabolites at the structural level, with sensitivity and speed inconceivable a few years ago, is the major driving force in the omic fields. The development of electrospray and matrix-assisted laser desorption/ionization (MALDI) ionization techniques has decisively contributed to the many applications of this technology nowadays. Herein, we present and discuss omic concepts and strategies, as well as detail basic principles of MS. Applications and future perspectives of these approaches are focused in the reproductive medicine area. Review: The omic technologies propose global characterization of specific classes of target biomolecules of cellular systems as a strategy to achieve comprehensive understanding of biological functions. The genomics, aimed at performing the entire genetic sequencing of organisms, represented the seminal step towards the understanding of the complex logic that orchestrates the function of all organisms or the defects leading to diseases. But to express the phenotype, information needs to flow from DNA via carrier biomolecules through processes that are being addressed by new omic sciences such as the transcriptomics, proteomics, metabolomics, glycomics, lipidomics, and fluxomics. Mass spectrometry (MS) is nowadays the most powerful technique for the structural characterization of biomolecules, and has therefore become the central technique for the omic sciences. Using revolutionary ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), a wide range of biomolecules such as peptides, proteins, lipids and sugars are efficiently transferred in intact ionized forms to the gas phase for MS analysis. The development of ESI-MS and MALDI-MS has been awarded the Nobel Prize for Chemistry in 2002, rocketing the application of MS in the omic sciences. More recently, ambient ionization MS techniques, such as desorption electrospray ionization (DESI) and easy ambient sonic-spray ionization (EASI), have been developed for ionization in the open atmosphere, in a workup free and high throughput fashion directly from sample in their original environments. For the more complex samples, the coupling with separation techniques such as liquid chromatrography (LC) as well as the use of tandem MS (LC-MS/MS) has allowed comprehensive mixture characterization of major biomolecules. Conclusion: This manuscript describes recent advances of MS in the proteomics, metabolomics and lipidomics for biological sciences, and points out the relevant contributions that MS is likely to bring to fundamental and applied research in human and animal embryo biotechnologies.