Improving in vitro maturation and pregnancy outcome in cattle using a novel oocyte shipping and maturation system not requiring a CO2 gas phase.

The present work evaluated the benefit of a novel shipping and maturation medium (SMM) not requiring a CO2 gas for maturation and subsequent embryonic development of slaughterhouse and ovum pickup (OPU) bovine cumulus-oocyte complexes (COCs). Four experiments were conducted. In experiment 1, COCs were maturated for 18 hours in SMM and then incubated for 6 hours in, or 24 hours in a conventional system (control). Experiment 2 compared maturation for 24 hours in SMM versus 24 hours in the control. Experiment 3 compared three different incubation temperatures (37 °C, 38 °C, and 38.5 °C) for COCs maturation in SMM. In experiment 4, COCs obtained from 166 OPU sessions (representing two dairy and two beef breeds) in two locations (Wisconsin and California) were matured in SMM or control and evaluated relative to embryo production and pregnancy rates. Frozen semen was used for all experiments. The results for experiment 1 showed that the blastocyst rate and total embryo production rate (TE, Day-7 morulae plus all blastocysts) were higher for SMM than those in the control. However, no differences were observed for cleavage rate or blastocyst stage. In experiment 2, the blastocyst rate and TE were higher for SMM than those in the control; however, there was no difference for cleavage rate, total cell number, blastocyst stage. In experiment 3, the cleavage rate was similar, but the blastocyst rate and TE were greater for 38.5 °C than those for 38.0 °C and 37.5 °C. For experiment 4, Wisconsin OPU-derived COCs had a greater cleavage rate, blastocyst rate, TE, and blastocyst stage for SMM versus control. There were no breed effects. For the California trial, OPU-derived COCs matured in SMM had similar cleavage and pregnancy rates at Day 35 but greater blastocyst rates and transferred embryos per session than the control, which resulted in 2.2 more pregnancies per OPU session. Holstein COCs had superior embryonic development but similar pregnancy compared with Jersey. We conclude that COCs matured in SMM had greater oocyte competence than the control. Also, maturation at 38.5 °C in SMM was optimal for embryonic development. In summary, SMM resulted in greater embryonic development, similar pregnancy rates, but higher pregnancies per OPU session than the conventional maturation system.

Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation.

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.

Molecular control of mitochondrial function in developing rhesus monkey oocytes and preimplantation-stage embryos.

The mitochondrion undergoes significant functional and structural changes, as well as an increase in number, during preimplantation embryonic development. The mitochondrion generates ATP and regulates a range of cellular processes, such as signal transduction and apoptosis. Therefore, mitochondria contribute to overall oocyte quality and embryo developmental competence. The present study identified, for the first time, the detailed temporal expression of mRNAs related to mitochondrial biogenesis in rhesus monkey oocytes and embryos. Persistent expression of maternally encoded mRNAs was observed, in combination with transcriptional activation and mRNA accumulation at the eight-cell stage, around the time of embryonic genome activation. The expression of these transcripts was significantly altered in oocytes and embryos with reduced developmental potential. In these embryos, most maternally encoded transcripts were precociously depleted. Embryo culture and specific culture media affected the expression of some of these transcripts, including a deficiency in the expression of key transcriptional regulators. Several genes involved in regulating mitochondrial transcription and replication are similarly affected by in vitro conditions and their downregulation may be instrumental in maintaining the mRNA profiles of mitochondrially encoded genes observed in the present study. These data support the hypothesis that the molecular control of mitochondrial biogenesis, and therefore mitochondrial function, is impaired in in vitro-cultured embryos. These results highlight the need for additional studies in human and non-human primate model species to determine how mitochondrial biogenesis can be altered by oocyte and embryo manipulation protocols and whether this affects physiological function in progeny.

Hand-made cloning approach: potentials and limitations.

Two major drawbacks hamper the advancement of somatic cell nuclear transfer in domestic animals. The first is a biological problem that has been studied extensively by many scientists and from many viewpoints, including the cell, molecular and developmental biology, morphology, biochemistry and tissue culture. The second is a technical problem that may be responsible for 50% or more of quantitative and/or qualitative failures of routine cloning experiments and is partially the result of the demanding and complicated procedure. However, even the relatively rare documented efforts focusing on technique are usually restricted to details and accept the principles of the micromanipulator-based approach, with its inherent limitations. Over the past decade, a small alternative group of procedures, called hand-made cloning (HMC), has emerged that has the common feature of removal of the zona pellucida prior to enucleation and fusion, resulting in a limited (or no) requirement for micromanipulators. The benefits of HMC are low equipment costs, a simple and rapid procedure and an in vitro efficiency comparable with or higher than that of traditional nuclear transfer. Embryos created by the zona-free techniques can be cryopreserved and, although data are still sparse, are capable of establishing pregnancies and resulting in the birth of calves. Hand-made cloning may also open the way to partial or full automation of somatic cell nuclear transfer. Consequently, the zona- and micromanipulator-free approach may become a useful alternative to traditional cloning, either in special situations or generally for the standardisation and widespread application of somatic cell nuclear transfer.

Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts.

The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts. In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation (n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1). medium only control; (2). activin (10 ng/ml); (3). epidermal growth factor (EGF) (10 ng/ml); (4). insulin 5 microg/ml and (5). GH (100 ng/ml). There was an increase (P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls. There was no significant difference between insulin and control groups. A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone. After thawing, embryos were cultured in five treatments groups as described above. Groups EGF and GH had higher (P<0.05) survival rates with a mean blastocyst survival of 95.0+/-1.5 and 93.1+/-3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3+/-3.4 and 62.0+/-3.2%, respectively). Thawed control blastocysts had a mean cell count of 52.7+/-3.3%. With the exception of insulin, all growth factors and GH tested showed higher (P<0.01) total cell numbers when compared to controls. In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.

The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts.

A total of 678 bovine blastocysts, which had been produced by in vitro maturation, fertilization, and culture, were placed into plastic straws and were vitrified in various solutions of ethylene glycol (EG) + polyvinylpyrrolidone (PVP). Part of the straw was loaded with TCM199 medium + 0.3 M trehalose as a diluent; the diluent portions of the straw were prefrozen to either -30 or -196 degrees C. Then, the embryos suspended in the vitrification solution were pipetted into the balance of the straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were warmed for 3 s in air and 20 s in a water bath at 39 degrees C and then agitated to mix the diluent and cryoprotectant solution for 5 min followed by culture in TCM199 + 10% FCS + 5 + microg/ml insulin + 50 microg/ml gentamycin sulfate for 72 h. Variables that were examined were the time of exposure to EG prior to vitrification, the PVP concentration, and the temperature of exposure to EG + PVP prior to vitrification. Survival and hatching rates of the blastocysts exposed to 40% EG in four steps at 4 degrees C were higher than those of embryos exposed in two steps (81.3 +/- 4.3% and 80.2 +/- 3.4% vs 67.6 +/- 4.5% and 71.5 +/- 4.7%, respectively; P < 0.05). The same indices were superior following vitrification-thawing of the blastocysts in 40% EG + 20% PVP than it was in 40% EG + 10% PVP (76.1 +/- 5.5% vs 63.7 +/- 1.8%; P < 0.05; and 61.6 +/- 6.0% vs 70.5 +/- 4.7%; P < 0.01, respectively). Exposure to the vitrification solution (40% EG + 20% PVP) at higher temperatures (37.5 degrees C vs 4 degrees C) reduced both survival and hatching rates (45.8 +/- 6.9% vs 83.9 +/- 4.4% and 41.5 +/- 1.8% vs 64.0 +/- 4.7%, respectively; P < 0.001). These results indicate that blastocysts vitrified after prefreezing the diluent portions of the straws do favor developmental competence of in vitro produced embryos.

Improvement of the culture conditions for in vitro production of cattle embryos in a portable CO2 incubator.

The effects of different concentrations of growth hormone (GH) on in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocyte/embryos in CR1aa or CR2aa media using a simple CO2 incubator were investigated. The IVM/IVF/IVC of oocytes were carried out in the presence of 0, 50, 100 and 200 ng/ml GH in the medium. The proportion of metaphase II oocytes was significantly higher (p < 0.05) in 200 ng/ml compared with 0 ng/ml GH in CR1aa medium (59 versus 85%, respectively), but this effect was not observed under CR2aa. Higher concentrations of GH yielded lower rates of unfertilized ova and thus superior cleavage rates (36.5 +/- 0.2 and 63.5 +/- 2.0% versus 17.5 +/- 0.2 and 82.5 +/- 1.5% or 40.4 +/- 0.6 and 59.6 +/- 1.4% versus 16.6 +/- 1.2 and 83.4 +/- 6.2% for 0 and 200 ng/ml GH in portable or ordinary incubator, respectively) in CR1aa. This dose-dependent effect was also observed in the percentages of transferable embryos, although not statistically different (17.2 +/- 1.7 versus 27.3 +/- 1.8% and 16.6 +/- 3.1 versus 26.0 +/- 1.4%, for 0 versus 200 ng/ml GH in portable and ordinary incubator, respectively). In contrast to the CR1aa, different concentrations of GH in CR2aa medium did not increase either fertilization or cleavage rates. In fact, higher concentrations of GH in this medium negatively affected the rate of transferable embryos. Hence, percentages of transferable embryos obtained in the portable incubator under 0 or 50 ng/ml GH were higher (p < 0.05) compared with those obtained in 100 or 200 ng/ml GH (35.4 +/- 5.7 or 40.5 +/- 5.4% versus 22.4 +/- 2.4 or 15.5 +/- 2.1%, respectively). There was however, no significant difference in the rate of transferable embryos in an ordinary incubator employing CR2aa medium, but the trend was more or less similar to that observed in the portable incubator. Despite the fact that relatively fewer oocytes were employed for the culture in the ordinary incubator, overall results observed employing the simple portable CO2 incubator were within the range of those obtained in an ordinary incubator: implying that the simple portable incubator can effectively be employed for the in vitro production of bovine embryos under field conditions.

Bovine blastocysts obtained from reconstructed cytoplast and karyoplasts using a simple portable CO2 incubator.

To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO(2) incubator. CO(2) and O(2) gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO(2) (2-5%) and O(2) (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5 degrees C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O(2), CO(2), and air pressure.