A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has been described as a technique to obtain transgenic offspring in mice. However, this approach has had limited success in domestic animals due to poor embryo development after ICSI. A first experiment was designed to improve embryo development comparing ICSI-mediated gene transfer with or without chemical activation (CA) in the ovine species. In the second experiment, ICSI-mediated gene transfer assisted by CA was used in porcine, feline, equine, and bovine species. Maturation and culture were done by standard procedures. Semen was collected by artificial vagina in ovine and bovine species. In pigs, ejaculates were obtained using the gloved-hand method, and in feline and equine species, sperm were obtained from epididymides. Samples were frozen by standard means. Thawed spermatozoa were washed twice in Na citrate at 2.8% with 100 µm EDTA at 495g for 5 min and resuspended in Na citrate with 0.5 µg of pCX-EGFP/million spermatozoa for 5 min at 0�C. The pCX-EGFP plasmid contained the egfp gene expressed under chimerical CMV-IE-chicken β-actin promoter control. Sperm cells were immediately injected into the metaphase II oocyte and CA was induced by incubation in TALP-HEPES with 5 µm ionomycin for 4 min, cultured in TCM199 for 3 h, and transferred to a droplet of 1.9 mm 6-dimethylaminopurine (DMAP) for 3 h. During the in vitro culture, exposure to blue light (488 nm) was performed to determine the percentage of green embryos, mosaic expression, and earliest stage of egfp expression. Fluorescence in situ hybridization analysis was performed labeling pCX-EGFP plasmid by nick translation for use as a probe. Statistical analysis was done by chi square. In ovine species, development to blastocyst stage (0/88 v. 3/86; P > 0.05) and number of green embryos (24/88 v. 39/86; P < 0.05) were greater with CA. The egfp expression in ovine embryos assisted by CA began at the 2- (7/39), 4- (9/39), or 8-cell (23/39). However, the expression in ovine embryos without CA occurred only at the 8-cell stage (24/24) stage. In porcine, bovine, feline, and equine species, green embryos were detected at a high proportion (33/55, 10/44, 9/35, and 5/17, respectively), and the percentage of fluorescent blastocysts was 2.3, 2.9, and 9.1% for ovine, feline, and bovine species, respectively. The egfp expression in porcine and feline embryos started at the 2-cell stage (36 and 22%, respectively), whereas it began in bovine and equine embryos at the 4-cell stage (9 and 40% respectively). All species showed a high frequency of mosaic expression (range 60-85%), and the preliminary FISH analysis demonstrated a variable number of integration events in porcine embryos. To our knowledge, this is the first report of exogenous DNA expression in feline and equine embryos. These results suggest that the CA accelerates and increases the pCX-EGFP expression in ovine embryos in agreement with previous studies that have shown earlier expression of genes for parthenogenetic and cloning embryos, both assisted by CA. In conclusion, ICSI-mediated gene transfer assisted by CA can be used to obtain exogenous gene-expressing embryos in domestic species with potential scientific and commercial interests.

Metabolomics reveals early predictors of blastocyst formation in equine ICSI-derived embryos.

Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity among the different members of the genus Equus, the domestic horse can be used to optimize cryopreservation and embryo production protocols for future application in wild equids. We will give this hypothesis a scientific underpinning by listing successful applications of epididymal sperm freezing, embryo freezing, intracytoplasmic sperm injection, oocyte vitrification and somatic cell nuclear transfer in domestic horses. Some ART fertilization methods may be performed with semen of very low quality or with oocytes obtained after the death of the mare.

Progress in assisted reproduction in the horse has been rapid over the last 10 years.Efficient methods for oocyte recovery from live mares have generated clinical interest in methods for in vitro fertilization (IVF).The conventional method of IVF as used in the bovine has proved to be inefficient in the horse.Therefore, a method for more efficient production of equine zygotes has been developed -the intracytoplasmic sperm injection (ICSI).The aim of our investigation was to establish this method in our laboratory and to test its efficiency in generating developmentally competent equine embryos.Altogether, 146 COCs with different cumulus morphology were recovered.The polar body extrusion rate after IVM was between 48 and 64 % depending on the cumulus morphology and the maturation time.The cleavage rate after ICSI was between 40% and 47%.Under the chosen in vitro conditions, we could obtain 16 cell stage embryos and morulae on day 5 after ICSI.

Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT)

The objective of this study was to test if intracytoplasmic sperm injection (ICSI)-mediated gene transfer was an effective method in the production of transgenic rabbit embryos. Rabbit sperm diluted in different media with various pH were treated by freezing without cryoprotectant, and their ability for DNA uptake was determined. In these experiments using production of transgenic rabbit embryos by ICSI, exogenous genes at three concentrations and of two conformation types were used. The rate of DNA association to the sperm seen by rhodamine-tagged DNA encoding green fluorescent protein (GFP) was 90.0%, 92.7%, 91.0%, 91.7%, and 92.3%, respectively in TCM199, DM, DPBS, CZB, and HCZB media. The DNA attachment to sperm was not affected by media pH within the range of 5.4-9.4 (p > 0.05). Expression of GFP first occurred at the 2-cell stage and continued to blastocyst formation. DNA concentration (between 5, 10, and 20 ng/μl) or conformation (linear and circular) had no effect on the production rate of transgenic embryos. These results indicated that genetically modified rabbit blastocysts can be efficiently produced by ICSI technique.

Equine embryos at the compacted morula and blastocyst stage can be obtained by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with frozen–thawed spermatozoa from semen of different fertilities

Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program

Advances in Holding and Cryopreservation of Equine Oocytes and Embryos

In vitro-produced equine embryos: Production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P < 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P < 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P < 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n = 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos.

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.

Transcriptomic profiling reveals similarities between equine IVF and ICSI embryos.

Intracytoplasmic sperm injection (ICSI) has been intensively used to examine the early events of gamete activation, but few studies have been reported for swamp buffalo. The first objective (Exp. 1) was to compare the developmental competence of oocytes after ICSI using either live or dead frozen-thawed spermatozoa. Matured oocytes were fertilized by ICSI using live (n = 148) or dead (n = 151) spermatozoa, followed by chemical activation using calcium ionophore (A23187) and cyclohexamide (CHX) in SOF medium. In vitro fertilization (n = 149) served as thecontrol. Cleavage rate was recorded on Day 2 and blastocyst formation rate was evaluated on Day 7. The second objective (Exp. 2) was to examine the effects of ICSI and activation regime on the decondensation of buffalo spermatozoa. A total of 148 matured oocytes were subjected to ICSI. The sperm-injected oocytes (n = 87) were then activated using the activation protocol as described in Exp. 1. The ICSI oocytes without activation (n = 61) and sham-injected oocytes with activation (n = 35) were used as controls. Nuclear changes of presumptive zygotes were mor- phologically evaluated for pronuclear formation using 4′,6-diamidino-2-phenylindole procedure and epifluorescent microscopy at 18 h post-ICSI. Statistical differences were determined among the groups using chi-square test. In Exp. 1, the results showed that the percentages of cleavage and blastocyst formation rate were 79.7, 77.0, and 41.6% and 33.8, 30.5, and 14.8% in the live sperm, dead sperm, and IVF groups, respectively. Embryo development rates did not significantly differ between ICSI groups; however, these rates were significantly higher than in the IVF group (P &lt; 0.05). In Exp. 2, the pronuclear formation rate was significantly higher in the ICSI with chemical (70.1%) and sham injection with chemical (60.6%) groups than in the ICSI without chemical group (3.2%; P &lt; 0.01). However, most of the presumptive zygotes with pronuclear formation from the ICSI with chemical activation group showed only intact sperm heads instead of the full male pronuclear formation. Our study suggests that the chemical activation directly affected the female pronuclear formation and embryo development but that it was not associated with the male pronuclear formation. It is postulated that ICSI oocytes that developed to cleavage and blastocyst stages underwent parthenogenesis after chemical activation. This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).