Oocyte vitrification induces loss of DNA methylation and histone acetylation in the resulting embryos derived using ICSI in dromedary camel.

Oocyte cryopreservation has become an important component of assisted reproductive technology with increasing implication in female fertility preservation and animal reproduction. However, the possible adverse effects of oocyte cryopreservation on epigenetic status of the resulting embryos is still an open question. This study evaluated the effects of MII-oocyte vitrification on gene transcripts linked to epigenetic reprogramming in association with the developmental competence and epigenetic status of the resulting embryos at 2-cell and blastocyst stages in dromedary camel. The cleavage rate of vitrified oocytes following intracytoplasmic sperm injection was significantly increased compared with the control (98.2 ± 2 vs. 72.7 ± 4.1%, respectively), possibly due to the higher susceptibility of vitrified oocytes to spontaneous activation. Nonetheless, the competence of cleaved embryos derived from vitrified oocytes for development to the blastocyst and hatched blastocyst was significantly reduced compared with the control (7.7 ± 1.2 and 11.1 ± 11.1 compared with 28.1 ± 2.6 and 52.4 ± 9.9%, respectively). The relative transcript abundances of epigenetic reprogramming genes DNMT1, DNMT3B, HDAC1, and SUV39H1 were all significantly reduced in vitrified oocytes relative to the control. Evaluation of the epigenetic marks showed significant reductions in the levels of DNA methylation (6.1 ± 0.3 vs. 9.9 ± 0.5, respectively) and H3K9 acetylation (7.8 ± 0.2 vs. 10.7 ± 0.3, respectively) in 2-cell embryos in the vitrification group relative to the control. Development to the blastocyst stage partially adjusted the effects that oocyte vitrification had on the epigenetic status of embryos (DNA methylation: 4.9 ± 0.4 vs. 6.2 ± 0.6; H3K9 acetylation: 5.8 ± 0.3 vs. 8 ± 0.9, respectively). To conclude, oocyte vitrification may interfere with the critical stages of epigenetic reprogramming during preimplantation embryo development.

Pregnancy and Calving Rates of Cloned Dromedary Camels Produced by Conventional and Handmade Cloning Techniques and In Vitro and In Vivo Matured Oocytes.

Despite practical implication of cloning in camelids, its broad application has been hampered by technical and biological problems. Method of somatic cell nuclear transfer (SCNT) and oocyte competence are the principal technical and biological factors, respectively, that determine the cloning efficiency. This study, therefore, investigated differential contributions of two SCNT methods [modified handmade cloning (mHMC) vs. conventional (cNT)] and two recipient oocyte sources [abattoir-derived (Vitro) vs. FSH-stimulated (Vivo)] on the efficiency of dromedary camel cloning. The mHMC method supported similar rates of fusion, cleavage, and total blastocyst development, compared to conventional NT (cNT) (94, 89.1, and 68.5% vs. 78.9, 92, and 73.5%, respectively) when Vivo oocytes are used. However, using Vitro oocytes, mHMC supported significantly higher rates for these criteria, except for the cleavage, compared to cNT (95.5, 76.2, 25.2% vs. 75.3, 76.7, and 13.9%, respectively). A total of seven clones were born from mHMC/Vitro (four calves), mHMC/Vivo (one calf), cNT/Vitro (one calf), and mHMC/Vivo&Vivo (one calf)-derived embryos with overall efficiencies of 31.9, 26.6, 20, and 30% for initial pregnancy, 10.6, 6.6, 7.5, and 5% for development to term, and 8.5, 6.6, 2.5, 5% for development to weaving, respectively. To conclude, the quality of recipient oocyte greatly impacts cloning efficiency in vitro with no apparent carrying over effect on cloning efficiency in vivo, but the efficiency of SCNT method may compensate for the initial poor oocyte competence during in vitro and in vivo development of cloned embryos. The introduced mHMC could be a superior alternative to conventional method for simple, fast, and efficient production of cloned offspring in camelids.

Platelet rich plasma efficiently substitutes the beneficial effects of serum during in vitro oocyte maturation and helps maintain the mitochondrial activity of maturing oocytes.

Platelets contain most of the potent mitogenic factors present in serum and follicular fluid and intraovarian injection of autologous platelet rich plasma (PRP) was shown to improve ovarian function and development of preantral follicles. This study evaluated the effect of PRP on caprine oocyte maturation in vitro and subsequent fertilization and embryonic development. Cumulus oocyte complexes were cultured in a maturation medium supplemented with (1) fetal bovine serum (FBS, control), (2) PRP, extracted from healthy female goats, (3) polyvinyl alcohol (PVA), and (4) PVA plus PRP (PVA-PRP). The degree of cumulus expansion was scored, and denuded oocytes were used for assessment of nuclear maturation, mitochondrial activity, lipid content, redox status, yield and quality of in vitro embryo development, and cryosurvival of the resulting blastocysts. PRP supported the same beneficial effects of FBS on cumulus expansion, nuclear maturation, in vitro developmental competence of oocytes, and survival of vitrified-warmed blastocysts. Moreover, PRP protected oocytes from undesirable effects FBS exerted on the mitochondrial activity and intracytoplasmic lipid content of maturing oocyte. Although PVA could support the same beneficial effects of neither FBS nor PRP on oocyte maturation, its combined addition with PRP improved the yield and quality of oocyte maturation at rates closely similar to PRP. PRP efficiently substitutes beneficial effects of serum during in vitro oocyte maturation and helps maintain the mitochondrial activity of maturing oocytes.

Effect of macromolecule supplement on nuclear and cytoplasmic maturation, cryosurvival and in vitro embryo development of dromedary camel oocytes.

The current evaluation of oocyte vitro maturation (IVM) media has progressed toward more defined conditions in human and livestock. In this study, the replacement of fetal calf serum (FCS) with bovine serum albumin (BSA) and polyvinyl alcohol (PVA) was evaluated during IVM in dromedary camel. Nuclear maturation rates in presence of FCS and PVA were comparable (81.6 ±â€¯1 and 75.5 ±â€¯5%, respectively). BSA, whether used alone or in combination with FCS, significantly reduced nuclear maturation (51.6 ±â€¯3.9 and 54.6 ±â€¯1.1%, respectively), compared to FCS and PVA. BSA also increased the rates of chromosome aberrations compared to FCS and PVA (25.7 ±â€¯7.4, 8.8 ±â€¯2.3 and 6.0 ±â€¯2.0%, respectively). IVM macromolecule differentially affected morphological aspects of cumulus expansion and FCS promoted the highest dissociation of cumulus cells, compared to all the other groups. FCS significantly increased mean lipid intensity of oocytes compared to BSA, FCS-BSA and PVA which could explain the lower cryo-survival of oocytes matured in presence of FCS compared to BSA and PVA (56.1 ±â€¯5.2, 91.0 ±â€¯19.5, and 87.8 ±â€¯6.7%, respectively). Mitochondrial activity was not affected by macromolecules, but oocytes cultured with PVA had the best redox status, compared to other IVM groups. Cleavage was not affected by IVM macromolecule, but FCS promoted significantly higher rate of morula development (51.6 ±â€¯5.2 vs. 33.6 ±â€¯2.9% for PVA) and blastocyst development (36.8 ±â€¯1.4 vs. 20.5 ±â€¯2.0% for BSA). Although adding FCS during IVM supported highest hatching rate of the resulting blastocysts, differential cell number showed no long lasting effect of IVM macromolecules on blastocyst quality. Obtained results suggest the possibility to switch from undefined to more defined IVM systems for efficient in vitro maturation and subsequent vitrification of dromedary camel oocytes. Keywords: camel, oocyte maturation, protein supplement, cryosurvival.

Diverse patterns of cumulus cell expansion during in vitro maturation reveal heterogeneous cellular and molecular features of oocyte competence in dromedary camel.

During in vitro maturation (IVM), the degree and pattern of cumulus cells expansion vary between cumulus oocyte complexes (COCs). This study investigated the relationship between expansion morphology of cumulus cells with the oocyte maturation quality in dromedary camel. Following IVM for 30-32 h, COCs were classified into four distinct groups according to the morphological features of COCs (adherent vs. non-adherent to the bottom of culture dish) and cumulus cells (compact vs. expanded vs. dissociated). The predominant morphology was adherent/dissociated (45.6 ±â€¯7.1%), followed by adherent/compact (31.2 ±â€¯6.5%), and then non-adherent/expanded (13.1 ±â€¯2.8%) and non-adherent/compact (10.2 ±â€¯2.4%). The adherent/dissociated morphology was correlated with the best oocyte quality in terms of MII-maturation (90.6 ±â€¯5.0%), degeneration (22.4 ±â€¯5.3%), reactive oxygen species (2.5 ±â€¯0.5 arbitrary units), mitochondrial potential (12.9 ±â€¯2.4 orange/green fluorescence intensity ratio), zona dissolution time (46 s), peripheral distribution of cortical granules (92%), and also cleavage and blastocyst development (81.3 ±â€¯5.2 and 48.7 ±â€¯7.2%, respectively). In contrast, adherent/compact morphology was correlated with the lowest oocyte competence when examined for the aforementioned criteria (28.1 ±â€¯5.3%, 62.7 ±â€¯9.2%, 6.0 ±â€¯0.8, 2.5 ±â€¯1.3, 102 s, 36%, and 25.7 ±â€¯4.5 and 11.6 ±â€¯5.1%, respectively). Non-adherent COCs, either expanded or compact, were correlated with an intermediate oocyte competence compared to the two extreme groups. In short, diverse pattern of cumulus expansion reveal heterogeneous cellular and molecular features associated with in vitro maturation capacity in camel. Cumulus expansion morphology can be used as a non-invasive predictive marker of oocyte competence to optimize assisted reproductive technologies in camels.

Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes.

Recent accomplishments in the field of somatic cell nuclear transfer (SCNT) hold tremendous promise to prevent rapid loss of animal genetic resources using ex situ conservation technology. Most of SCNT studies use viable cells for nuclear transfer into recipient oocytes. However, preparation of live cells in extreme circumstances, in which post-mortem material of endangered/rare animals is improperly retained frozen, is difficult, if not impossible. This study investigated the possibility of interspecies-SCNT (iSCNT) in Asiatic cheetah (Acinonyx jubatus venaticus), a critically endangered subspecies, using nuclei derived from frozen tissue in absence of cryo-protectant at -20 °C and in vitro matured domestic cat oocytes. No cells growth was detected in primary culture of skin and tendon pieces or following culture of singled cells prepared by enzymatic digestion. Furthermore, no live cells were detected following differential viable staining and almost all cells had ruptured membrane. Therefore, direct injection of donor nuclei into enucleated cat oocytes matured in vitro was carried out for SCNT experiments. Early signs of nuclear remodeling were observed as early as 2 h post-iSCNT and significantly increased at 4 h post-iSCNT. The percentages of iSCNT reconstructs that cleaved and developed to 4-16 cell and morula stages were 32.3 ± 7.3, 18.2 ± 9.8 and 5.9 ± 4.3%, respectively. However, none of the iSCNT reconstructs developed to the blastocyst stage. When domestic cat somatic and oocytes were used for control SCNT and parthenogenetic activation, the respective percentages of oocytes that cleaved (51.3 ± 13.9 and 77.3 ± 4.0%) and further developed to the blastocyst stage (11.3 ± 3.3 and 16.8 ± 3.8%) were comparable. In summary, this study demonstrated that enucleated cat oocytes can partially remodel and reactivate non-viable nuclei of Asiatic cheetah and support its reprogramming back to the embryonic stage. To our knowledge, this is the first report of iSCNT in cheetah using non-viable frozen cells.

Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts.

BACKGROUND: This study describes the generation and analysis of the transcriptional profile of bovine inner cell mass (ICM) and trophectoderm (TE), obtained from in vivo developed embryos by using a bovine-embryo specific array (EmbryoGENE) containing 37,238 probes. RESULTS: A total of 4,689 probes were differentially expressed between ICM and TE, among these, 2,380 and 2,309 probes were upregulated in ICM and TE tissues, respectively (P ≤ 0.01, FC ≥ 2.0, FDR: 2.0). Ontological classification of the genes predominantly expressed in ICM emerged a range of functional categories with a preponderance of genes involved in basal and developmental signaling pathways including P53, TGFß, IL8, mTOR, integrin, ILK, and ELF2 signalings. Cross-referencing of microarray data with two available in vitro studies indicated a marked reduction in ICM vs. TE transcriptional difference following in vitro culture of bovine embryos. Moreover, a great majority of genes that were found to be misregulated following in vitro culture of bovine embryos were known genes involved in epigenetic regulation of pluripotency and cell differentiation including DNMT1, GADD45, CARM1, ELF5 HDAC8, CCNB1, KDM6A, PRDM9, CDX2, ARID3A, IL6, GADD45A, FGFR2, PPP2R2B, and SMARCA2. Cross-species referencing of microarray data revealed substantial divergence between bovine and mouse and human in signaling pathways involved in early lineage specification. CONCLUSIONS: The transcriptional changes occur during ICM and TE lineages specification in bovine is greater than previously understood. Therefore, this array data establishes a standard to evaluate the in vitro imprint on the transcriptome and to hypothesize the cross-species differences that allow in vitro acquisition of pluripotent ICM in human and mice but hinder that process in bovine.

A Novel Method of Somatic Cell Nuclear Transfer with Minimum Equipment.

Somatic cell nuclear transfer (SCNT) is an exceptional experimental biology technique with an arguably great contribution to our current understanding of developmental plasticity. Many students and young researchers are interested in taking advantage of SCNT virtues in their experiments but the cost of micromanipulation microscopes, intensive training programs, and also the sophisticated process of SCNT may dissuade them from entering this amazing field of science. Here, we describe the details of a streamlined manual method of SCNT that can be performed using very basic equipment found in every embryology laboratory: the Pasteur pipette and stereomicroscope. The overall method introduced is very simple and a person with no previous experience in cloning can learn and adopt the basic routines of this technique independently.

Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 on the efficiency of a manual method of oocyte enucleation using a pulled Pasteur pipette.

The present study aimed to facilitate widespread application of a previously described manual method of somatic cell nuclear transfer (SCNT) by investigating the effects of demecolcine (a microtubule-depolymerizing chemical), cytochalasin-B (a microfilament-depolymerizing chemical: 2.5µg/ml for 15min) and MG-132 (a proteasome inhibitor chemical) on the (i) incidence of cytoplasmic protrusion of MII chromosomes, (ii) improvement of manual oocyte enucleation, and (iii) in vitro and in vivo developmental competence of SCNT embryos in the goat. Following in vitro maturation, around 65% of goat oocytes contained a characteristic cytoplasmic protrusion of MII-chromosomes. Treatment with demecolcine (0.4µg/ml for 30min) significantly increased this rate to 92.2±4.5%. Treatment with MG-132 (2µM for 30min) could not improve this rate when used alone (61.4±11.5%), but when combined with demecolcine (86.4±8.1%). Treatment with cytochalasin-B completely suppressed this rate whenever used, either alone (7.7±5.1%) or in combination with demecolcine (3.9±1.3%). In a direct comparison, there was no significant difference in quantity and quality of embryos propagated by the manual vs. micromanipulation-based methods of SCNT (cleavage: 85.3±4.5 vs. 89.5±8.9%, blastocyst: 19.5±4.3 vs. 24.3±4.4%, grade 1 and 2 blastocyst: 33.8±7.1 vs. 29.5±6.3%, total cell count: 125±11.1 vs. 122±10.5, respectively). Furthermore, development to live kids at term was not significant between the two SCNT methods. From both technical and economical points of view, the overall in vitro and in vivo efficiency of this manual method of SCNT proved it a simple, fast and efficient alternative for large scale production of cloned goats.