The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development.

Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24 h after IVM, respectively) nor the accelerated polar body emission (at 18 h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide.

Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.

Xenooplasmic transfer between buffalo and bovine enables development of homoplasmic offspring.

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line.

Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle.

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear-cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.

Demecolcine effects on microtubule kinetics and on chemically assisted enucleation of bovine oocytes.

This study aimed to evaluate the effect of demecolcine, a microtubule-depolymerizing agent, on microtubule kinetics; to determine the best concentration of demecolcine as a chemically assisted enucleation agent in metaphase I (MI) and metaphase II (MII) bovine oocytes, and to evaluate the embryonic development after nuclear transfer (NT) using chemically assisted enucleation of recipient oocytes. Oocytes in vitro matured for 12 h (MI) and 21 h (MII) were exposed to several concentrations of demecolcine and evaluated for enucleation or membrane protrusion formation. Demecolcine concentration of 0.05 microg/mL produced the highest rates of enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%), and was employed in the following experiments. Demecolcine effect was seen as early as 0.5 h after treatment, with a significant increase in the frequency of oocytes with complete microtubule depletion in MI (58.9%) and MII (21.8%) compared to initial averages at 0 h (27.4% and 1.9%, respectively). Microtubule repolymerization was observed when MII-treated oocytes were cultured in demecolcine-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). Chemically assisted enucleated oocytes were used as recipient cytoplasts in NT procedures to assess embryonic development. For NT, 219 of 515 oocytes (42.5%) formed protrusions and were enucleated, and reconstructed, resulting in 58 nuclear-transferred one-cell embryos. Cleavage (84.5%) and blastocyst development (27.6%) rates were assessed. In conclusion, demecolcine can be used at lower concentrations than routinely employed, and the chemically assisted enucleation technique was proven to be highly efficient allowing embryonic development in bovine.

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos.

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Use of strontium for bovine oocyte activation.

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50 mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+- and Mg2+-free TALP for 6 h), ionomycin+strontium (IS), and strontium+ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 microM for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to I, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+- and Mg2+-free TALP medium for 6 h.

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer.

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium.

Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1). to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2). to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10mM SrCl(2) for 5h) alone or in combination: ethanol+strontium (ES) and strontium+ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (D7). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P>0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P>0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 h, respectively; P<0.05) and strontium after 30 h (53.3%) was superior to 24 and 26 h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P>0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82+/-5.7 and 89.5+/-7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P>0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl(2) induces activation and parthenogenetic development in bovine oocytes.