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ABSTRACT: Fewer follicles and of variable size are found at any time on the mare ovary compared with other livestock species, also influenced by seasonal variation. This is reflected on the population of cumulus-oocyte complexes (COC) collected that is characterised by a high heterogeneity. Recovered immature oocytes presumably need different culture conditions. The main factors contributing to this high heterogeneity are the follicle diameter, the status of the cumulus oophorus, and the reproductive seasonality. Therefore, the aim of the present study was to assess the nuclear chromatin configuration and the oocytes-cumulus cell gap junction-mediated communication (GJC) functionality in COC of different origins because these parameters are indicative of the oocyte's metabolic state and should be taken into account when designing IVM strategies. The COC with compact (Cp) or expanded (Ex) cumulus oophorus were collected from follicles of different diameters (<1, 1-2, and >2cm) in October-November, January-February, and April-May. The GCJ functionality was assessed by Lucifer Yellow microinjection and chromatin configuration was evaluated by Hoechst and Lacmoid staining, after cumulus cells removal. Data were obtained from a total of 1003 oocytes and were analysed by chi-squared test. Overall, GJC functionality was impaired in the majority of Ex COC in each follicle category, even though a certain proportion of them had open GJC (% of Ex COC with open GJC was 39.7, 29.3, and 39.3 in <1, 1-2, and >2cm follicles, respectively). Moreover, the proportion of Ex COC with open GJC did not differ significantly between periods (% of Ex COCs with open GJC in October-November, January-February, and April-May was, respectively, 43.3, 28.6, and 41.7 in <1cm follicles; 45.5, 19.3, and 26.47 in 1-2cm follicles; 66.7, 50, and 16.7 in >2 follicles). On the contrary, the majority of Cp COC from follicles <1 and 1-2cm, showed open GCJ in October-November and April-May, whereas they decreased significantly in January-February. This tendency was not maintained in Cp COC from follicles >2cm, where GJC functionality did not differ significantly between periods (% of Cp COC with open GJC in October-November, January-February, and April-May was, respectively, 74.4, 35.7, and 75 in <1cm follicles; 73.8, 42.1, and 67.7 in 1-2cm follicles; 58.3, 58.3, and 68.6 in >2-cm follicles). Chromatin configuration analysis revealed that the highest proportion (23.9%) of oocytes with fibrillar chromatin was found in Cp oocytes from <1cm follicles, whereas the proportion of oocytes with fibrillar chromatin ranged from 5.4 to 12.5% in the other groups. Moreover, the increase in follicle diameter was generally associated with an increase of chromatin condensation in Cp COC. Interestingly, the chromatin configuration distribution did not differ significantly among seasons. Our data could be useful in setting up new in vitro cultural strategies aimed to improve horse-assisted reproductive technology efficiency as well as in the understanding of horse oocyte biology.
Reproduction Fertility and Development 12/2012; 25(1):241. · 2.11 Impact Factor
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ABSTRACT: Because of the limited availability of horse oocytes and their wide maturation window that leads to the existence of a subpopulation of oocytes with significantly longer maturation period, horse cloning would benefit from the possibility of also using metaphase I oocytes (Choi et al. 2009 Cloning Stem Cells). The scope of this work was to compare the developmental ability of cloned horse embryos constructed using oocytes in metaphase I (MI) and II (MII). Oocytes of slaughtered mares were matured for 27h in DMEM/F12 with 10% FCS, 1µLmL(-1) of insulin-transferrin-selenium (ITS), 1mM sodium pyruvate, 50ngmL(-1) of long-epidermal growth factor, 100ngmL(-1) of long-insulin-like growth factor-I, and 0.1IUmL(-1) each of FSH and LH, denuded of cumulus and enucleated using a zona-free method (Lagutina et al. 2005 Reproduction). Oocytes with a visible polar body were classified as MII and those with no polar body as potential MI. During enucleation, oocytes having a metaphase plate only were confirmed as MI; oocytes in anaphase and telophase were classified as MII. Adult skin fibroblasts of passages 2 to 10 were cultured in TCM 199/DMEM with 10% FCS and serum starved for 1 to 2 days before NT. Nuclear-transfer embryos were constructed after washing of zona-free oocytes in 400µgmL(-1) of phytohemagglutinin P and attachment of each to a single cell in HEPES-SOF by fusion with 2 direct-current (DC) pulses of 1.2kVcm(-1) applied for 30µs in fusion medium. One hour after fusion, embryos were activated by 5µM ionomycin for 4min, followed by culture in 5µgmL(-1) of cycloheximide and 1mM DMAP in SOFaa for 3h. Embryos were cultured in SOFaa in 5% CO(2) and 5% O(2) at 38.5°C. Half of the medium was renewed on Day 3 and replaced on Day 5 with DMEM/F12 with 5% FCS and 5% serum replacement. Cleavage was assessed 48h after activation and the rate of blastocyst formation was recorded at Day 8. The data were compared by chi-square test. The development ability of MI embryos assessed by cleavage and blastocyst formation was significantly lower than of MII embryos (Table 1). The obtained MI blastocysts were smaller than their MII counterparts. These data demonstrate that MI oocytes account for 20% of the total oocytes after 27h of maturation, have a lower developmental competence to form blastocysts after NT, and the blastocysts obtained are of smaller size and likely less viable. Therefore, the use of MI oocytes can only marginally improve the outcome of horse cloning.
Reproduction Fertility and Development 12/2012; 25(1):165. · 2.11 Impact Factor
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ABSTRACT: 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.
Reproduction in Domestic Animals 06/2012; 47 Suppl 3:2-11. · 1.36 Impact Factor
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ABSTRACT: In the buffalo, the use of embryo-based biotechnologies for breeding and genetic improvement is still very limited because multiple-ovulation embryo transfer delivers poor results compared with cattle and in vitro embryo production has been used mainly for research purposes. At present, very few reports are available on the transfer of in vitro-produced (IVP) and cryopreserved buffalo embryos. Therefore, the scope of this work was to perform a pilot study to evaluate the viability of frozen-thawed IVP embryos by nonsurgical embryo transfer to recipients in an IVF-embryo transfer program on a farm located on the north coast of Colombia, South America. Buffalo oocytes were recovered at the slaughterhouse from selected donors, matured in vitro for 18 to 20h in TCM-199+10% FCS and 0.5 IU of FSH and 0.5 IU of LH in 5% CO(2) at 38.5°C. Four different bulls were used for IVF. After thawing, the semen was separated on a Percoll(®) gradient and then diluted into SOF-IVF media supplemented with 1μgmL(-1) of heparin and phenylalanine. Presumptive zygotes were cultured in modified SOF supplemented with MEM amino acids for 6 days. Half of the medium was replaced on Day 4 and 6. Developing embryos were selected for freezing on Day 6 and 7. Grade 1 embryos were frozen at the blastocyst stage by slow cooling in 10% glycerol or 1.5M ethylene glycol. Recipients (heifers n=79 and uniparous cows n=17) were synchronized using the CIDR-Synch protocol: on Day 0, gonadotropin-releasing hormone was injected and a CIDR was inserted; on Day 7, prostaglandin F(2α) was administered; on Day 9, the CIDR was removed; on Day 11, a second injection of gonadotropin-releasing hormone was given; and on Day 17, the embryo was transferred. Each female received, nonsurgically, 1 or 2 embryos in the ipsilateral horn to the functional corpus luteum evaluated by ultrasonography. Pregnancies were evaluated by ultrasonography 30 days after transfer and confirmed by rectal palpation 30 days later. This work was performed in 2 successive experiments during the breeding seasons (January and December, respectively). Overall, 96 recipients were transferred, with 136 embryos obtaining 23 pregnancies (24.2%). There were no statistical differences in pregnancy rate between heifers and cows (25.3 vs 17.7%) and between single (n=56) and double (n=39) embryo transfers (21.4 vs 27.5%) by chi square test (P>0.05). To date, 4 females and 5 males have been born by spontaneous calving (1 stillborn male due to dystocia), 3 pregnancies have been aborted (13%) and 11 pregnancies are ongoing (>7 months). The pregnancy rate obtained in this study in farm conditions (24.2%) is lower than generally obtained with frozen IVP cattle embryos, but it is still a good result in buffalo, where even conventional AI provides a lower success rate as compared with cattle. Finally, this work demonstrates that in vitro embryo production can be successfully implemented in buffalo breeding programs for the exploitation of superior genetics.
Reproduction Fertility and Development 12/2011; 24(1):190-1. · 2.11 Impact Factor
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ABSTRACT: In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus-oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24h in defined culture medium supplemented with 0.1 IUmL(-1) of FSH in the presence or absence of 1μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0h) and after 12, 18, or 24h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843-1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P<0.05) and not significantly different from the time 0h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P<0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis.
Reproduction Fertility and Development 12/2011; 24(1):211. · 2.11 Impact Factor
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ABSTRACT: The genetic engineering of the pig genome for xenotransplantation studies requires the insertion of different transgenes to create multi-transgenic pigs. In order to simultaneously add more transgene in a single genetic insertion, we constructed a polycistronic vector using the F2A self-cleaving peptide. Moreover, this solution has the added advantages of preventing possible segregation during breeding of the animals and of guaranteeing an equimolar production of chosen transgenes. The scope of this work was the construction and validation of an ubiquitous F2A-bicistronic expression vector for human thrombomodulin (hTM) and human endothelial protein C receptor (hEPCR) genes in pig primary hCD55-GAL(-/-) cells to establish transgenic fibroblasts colonies, to be used for somatic cell nuclear transfer (SCNT) to generate pigs for xenotransplantation research. The expression vector consisted of pCAGGS promoter (CMV-IE+chicken β actin) followed by hEPCR-furinF2A-hTM coding sequence. The resulting expression cassette was inserted between 2 insulators obtained from the 5' MAR region of chicken lysozyme. Outside of this insulated structure, there is a loxable puromycin selection cassette. The resulting purified and linearized expression vector (pEFTM/Lgu I=5μg) was transfected into hCD55-GAL(-/-) primary fibroblasts (1×10(6)), using Nucleofector (Amaxa, Lonza, Cologne, Germany), in parallel for comparative purposes we cotransfected the 2 pCAGGS-monocistronic vectors for the same transgenes (hEPCR and hTM=1:3, 5μg). Transfected cells were selected with puromycin (1μgmL(-1)) for 15 days. After 8 days of selection, resistant colonies were picked up and expanded into 24-well plates for cryopreservation and analyses. Bicistronic transfection produced 20 clones and cotransfection only 8 clones that were analysed by Western blot (WB) and by immunocytochemistry (ICC) using polyclonal antibody anti-EPCR (1:250, R&D) and monoclonal antibody ab6980-Abcam (1:5000, Abcam, Cambridge, UK) in WB; polyclonal antibody RCR252 (1:100, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal antibody ab6980-Abcam (1:100, Abcam) for ICC. Seventeen bicistronic clones (85%) and 2 cotransfected monocistronic clones (25%) were positive for both transgenes using WB. After ICC analyses, only 11 bicistronic colonies (55%) and 1 cotransfected colony (12.5%) uniformly expressed the desired transgenes and were selected for SCNT. The pCAGGS promoter maintained its strong expression also using the hEPCR-FurinF2A-hTM coding sequence and this bicistronic solution permitted us to improve our results obtained with co-transfection. Availability of hEPCR(+) hTM(+) hCD55(+)-GAL(-/-) colonies will allow us to obtain a new transgenic background for future xenotransplantation projects.
Reproduction Fertility and Development 12/2011; 24(1):229-30. · 2.11 Impact Factor
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ABSTRACT: The aim of this study was the development of an alternative testing method based on human embryonic stem cells for prenatal developmental toxicity with particular emphasis on early neural development. To this purpose, we designed an in vitro protocol based on the generation of neural rosettes, representing the in vitro counterpart of the developing neural plate and neural tube, and we challenged this complex cell model with retinoic acid (RA), a well-known teratogenic agent. The cells were exposed to different concentrations of RA during the process of rosettes formation. Morphological and molecular parameters were evaluated in treated as compared with untreated cells to detect both cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with microarray Affymetrix platform and validated by quantitative real-time PCR for genes relevant to early neural development such as HoxA1, HoxA3, HoxB1, HoxB4, FoxA2, FoxC1, Otx2, and Pax7. The results obtained demonstrated that neural rosette forming cells respond to RA with clear concentration-dependent morphological, and gene expression changes remarkably similar to those induced in vivo, in the developing neural tube, by RA exposure. This strict correspondence indicates that the neural rosette protocol described is capable of detecting specific teratogenic mechanisms causing perturbations of early neural development and therefore represents a promising alternative test for human prenatal developmental toxicity.
Toxicological Sciences 09/2011; 124(2):370-7. · 4.65 Impact Factor
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ABSTRACT: Both preimplantation and full-term development of mouse somatic cell nuclear transfer (SCNT) embryos are significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183-189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083-1089). The present study was designed to examine the effect of TSA treatment on preimplantation and full-term development of bovine cloned embryos. To investigate the effect of TSA on bovine NT embryos development, we treated them with 50nM TSA during the first 10h after activation. Bovine NT-embryos were reconstructed using adult fibroblasts of 2 female donors (A and B) with significantly different in vitro cloning efficiency (respectively, 84/245; 34.3% v. 155/298; 52.1% blastocyst D7, P≤0.05, chi-square test). TSA treatment significantly improved blastocyst rate in A, however did not affect development in B (56.3% and 50.5%, respectively). The level of acetylated histone H3K9 10h after activation detected by anti-acH3K9 antibody was significantly increased after TSA-treatment in A (P≤0.05, Student's t-test) but did not change in B, thus demonstrating that the levels of histone acetylation in cloned embryos correlate with their in vitro developmental potential. To evaluate the long-lasting effect of TSA-treatment on the full-term development of cloned embryos, SCNT embryos derived from 4 female donor animals were reconstructed. 196 TSA-treated embryos at the blastocyst stage were transferred into 98 recipients and 2 calves (2%) were born. In the control group, 167 embryos were transferred into 141 recipients and 3 calves (2.1%) were born. Our data show that cell lines demonstrate different susceptibility to TSA that may affect reprogramming of the somatic genome with low level of acetylation resulting in higher in vitro embryo development. However, TSA does not improve overall cloning efficiency in cattle, measured as full-term development.
Reproduction Fertility and Development 01/2011; 23(1):127-128. · 2.11 Impact Factor
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ABSTRACT: In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at approximately 60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 +/- 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.
Theriogenology 05/2010; 74(4):557-62. · 1.96 Impact Factor
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ABSTRACT: 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.
Theriogenology 09/2009; 73(6):748-57. · 1.96 Impact Factor
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ABSTRACT: Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in several species by artificial insemination, although the success rate is often lower than with non-sexed semen. In horses, the problem was partially overcome with hysteroscopic insemination using sex-sorted fresh sperm. However, when sex-sorted frozen-thawed sperm were used the pregnancy rate was heavily reduced in comparison with non-sexed frozen-thawed semen. Because it has been demonstrated that in vitro assisted reproductive techniques, namely intracytoplasmic sperm injection (ICSI), has permitted live foals to be obtained using sperm with low fertility in the field, in this study we investigated the possibility of using ICSI with sexed-sorted frozen-thawed sperm for equine embryo production in vitro. Briefly, semen was collected from two Standardbred stallions of proven fertility (Stallions A, B), sorted using a MoFlo SX flow cytometer and frozen (Johnson LA and Welch GR 1999 Theriogenology 52, 1323-1341). 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 and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen conventionally in 10% of glycerol (Galli et al. 2002 Theriogenology 58, 713-715). Six embryos from sexed-sorted sperm were thawed and non-surgically transferred in naturally cycling synchronous recipient mares. Results are summarized in Table 1. Overall, 70 and 58 (stallion A, B) and 30 and 15 (stallion C, D) oocytes were injected with sex-sorted or control frozen-thawed sperm, respectively. Mean cleavage rates were 20.3% for sorted sexed sperm and 71.1% for control, showing a significantly lower cleavage rate for sexed sperm. This difference was reflected in the number of blastocysts obtained (4.7% v. 20.0%). From the 6 frozen-thawed embryos derived from sexed sperm, that were transferred, 4 pregnancies resulted. One pregnancy was lost around 21 days, a second was pharmacologically aborted, and two were maintained (one from male and one from female sorted semen are currently in the 11th month of gestation). In conclusion, this study demonstrates that ICSI with sex-sorted sperm can be used for producing equine blastocysts able to establish pregnancies at a high rate following embryo transfer. However, the overall efficiency of the system is limited due to the very low cleavage rate obtained with sexed-sorted frozen-thawed sperm.
Reproduction Fertility and Development 01/2009; 21(1):228-229. · 2.11 Impact Factor
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ABSTRACT: This paper summarizes the major advances in farm animal assisted reproduction in the last 20 years with particular attention to the contributions of the authors. A main emphasis is on the biology of the oocyte, including a description of methods for isolation of developing follicles and culture of the corresponding oocytes. Milestones that have led to optimization of procedures for maturation of fully grown oocytes, fertilization, intracytoplasmic sperm injection and embryo culture in sheep, cattle, pigs and horses are described. The current status of nuclear transfer, cloning and embryonic stem cell generation and culture is also summarized for all major farm animal species. It is concluded that the manipulation of early development in farm animals is of crucial importance for agricultural purposes and that reproductive biotechnologies in farm animals are expected to play an increasing role in the next decades due to the growing demand for agricultural products from the emerging economies worldwide. In the biomedical field large animals represent increasingly important research models especially in the stem cell field for creating genetically modified animals for specific purposes. Finally, the successful translation of large animal research in the applied context requires solid science, long-term resource commitment from involved institutions, and vision, dedication and entrepreneurial skills from the scientists involved.
Reproduction in Domestic Animals 07/2008; 43 Suppl 2:1-7. · 1.36 Impact Factor
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ABSTRACT: 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.
Animal Reproduction Science 04/2007; 98(1-2):39-55. · 1.75 Impact Factor
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ABSTRACT: 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.
Reproduction (Cambridge, England) 12/2006; 132(5):759-69. · 3.09 Impact Factor
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ABSTRACT: Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.
Molecular Reproduction and Development 10/2005; 72(1):77-87. · 2.53 Impact Factor
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ABSTRACT: 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.
Journal of Virological Methods 09/2005; 127(2):168-70. · 2.01 Impact Factor
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ABSTRACT: Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and “missing” signals were assumed to be “false negatives,” 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes. © 2005 Wiley-Liss, Inc.
Molecular Reproduction and Development 08/2005; 72(1):77 - 87. · 2.53 Impact Factor
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ABSTRACT: 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.
Cloning and Stem Cells 02/2005; 7(3):154-66. · 2.66 Impact Factor
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ABSTRACT: Zygote arrest 1 (Zar1) is an ovary-specific maternal factor that plays an essential role during the oocyte-to-embryo transition in mouse. In this species, Zar1 expression is strictly limited to the oocyte, the zygote and, at a lower level, the 2-cell embryo. Aim of the present study was to analyze the presence and the expression pattern of the Zar1 ortholog in bovine tissues and embryos. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis was performed in a panel of bovine tissues, in oocytes and pre-implantation in vitro produced embryos. The results demonstrated that a Zar1 ortholog is present in cattle. In the adult, the gene is expressed in ovary, testis, muscle, and myocardium. The gene is also expressed in the oocyte, the zygote, and in all the stages of embryonic development until blastocyst formation. A semi-quantitative RT-PCR analysis revealed that Zar1 levels are constant through in vitro development with the exception of the 4-cell stage, when a significant increase is observed. The exposure of fertilized oocytes to the RNA polymerase II inhibitor alpha-amanitin was able to suppress this Zar1 increase indicating that transcription of this gene occurs at the 4-cell stage. Zar1 is conserved in cattle but has an expression pattern different from the mouse. In particular, Zar1 expression in the adult is not limited to the ovary and in the embryo is expressed well beyond the oocyte to embryo transition. Moreover, the identification of Zar1 transcription at the 4-cell stage represents the first characterization of one of the genes expressed in cattle embryos before the major onset of embryonic transcription.
Molecular Reproduction and Development 01/2005; 69(4):375-80. · 2.53 Impact Factor
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ABSTRACT: Zygote arrest 1 (Zar1) is an ovary-specific maternal factor that plays an essential role during the oocyte-to-embryo transition in mouse. In this species, Zar1 expression is strictly limited to the oocyte, the zygote and, at a lower level, the 2-cell embryo. Aim of the present study was to analyze the presence and the expression pattern of the Zar1 ortholog in bovine tissues and embryos. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis was performed in a panel of bovine tissues, in oocytes and pre-implantation in vitro produced embryos. The results demonstrated that a Zar1 ortholog is present in cattle. In the adult, the gene is expressed in ovary, testis, muscle, and myocardium. The gene is also expressed in the oocyte, the zygote, and in all the stages of embryonic development until blastocyst formation. A semi-quantitative RT-PCR analysis revealed that Zar1 levels are constant through in vitro development with the exception of the 4-cell stage, when a significant increase is observed. The exposure of fertilized oocytes to the RNA polymerase II inhibitor alpha-amanitin was able to suppress this Zar1 increase indicating that transcription of this gene occurs at the 4-cell stage. Zar1 is conserved in cattle but has an expression pattern different from the mouse. In particular, Zar1 expression in the adult is not limited to the ovary and in the embryo is expressed well beyond the oocyte to embryo transition. Moreover, the identification of Zar1 transcription at the 4-cell stage represents the first characterization of one of the genes expressed in cattle embryos before the major onset of embryonic transcription. Mol. Reprod. Dev. 69: 375–380, 2004. © 2004 Wiley-Liss, Inc.
Molecular Reproduction and Development 11/2004; 69(4):375 - 380. · 2.53 Impact Factor
