Perspectives for artificial insemination and genomics to improve global swine populations

Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future

Reversible Disassembly of the Actin Cytoskeleton Improves the Survival Rate and Developmental Competence of Cryopreserved Mouse Oocytes

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method

Culture and cryopreservation of in vitro produced bovine embryos

This study evaluated procedures to improve culture and cryopreservation of in vitro produced bovine embryos. Successful in vitro development of early embryos is crucial for areas of biotechnology such as transgenics and cloning. The limiting factor in culturing in vitro matured and fertilized (IVM-IVF) embryos is the media in which the embryos develop in vitro. An optimal culture system which would support development equivalent to that found in vivo has yet to be found. If such a system were developed, studies could be conducted to elucidate the factors responsible for normal embryonic development and then apply these factors toward the development of defined media. The mitogenic capabilities of bovine (BVH) and rabbit (RVH) vitreous humor were tested on the in vitro development of IVM-IVF bovine embryos. Embryos were cultured from day 2, 3, 4, 5 or 6 until day 8 in different concentrations of BVH/RVH. Bovine embryos are inconsistent in their ability to develop in these fluids until the late morulae or early blastocyst stage. The activity of vitreous humor is not species-specific as bovine embryos developed in RVH as well or better than BVH. Blastocyst development was not improved with culture in BVH or RVH, although there is evidence to suggest that RVH enhances mean cell number in developing embryos. Delaying culture in BVH or RVH did improve mean cell number and blastocyst development. A simple and reliable method was developed to cryopreserve in vitro produced bovine embryos. The developmental capability of IVM-IVF derived morulae/early blastocysts was evaluated following different methods of diluting cryoprotectants after vitrification. One-step 0.1M or 0.3M sucrose dilution was optimal for removing the high concentrations of intracellular cryoprotectants in the vitrification medium as post-treatment survival was no different than culture controls. Step-wise dilution/gradual rehydration of vitrified embryos does not improve development following vitrification. Freezing and vitrification are equivalent for in vitro development of IVM-IVF bovine embryos following cryopreservation, although blastocyst development is approximately 1/2 the rate of their culture controls. These data are the first to indicate that IVM-IVF generated bovine embryos can be cryopreserved by vitrification

Characterization of the Altered Gene Expression Profile in Early Porcine Embryos Generated from Parthenogenesis and Somatic Cell Chromatin Transfer

<div><p>The <i>in vitro</i> production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from <i>in vitro</i> Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than <i>in vivo</i>–derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of <i>in vivo</i> (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The “eIF2 signalling”, “mitochondrial dysfunction”, “regulation of eIF4 and p70S6K signalling”, “protein ubiquitination”, and “mTOR signalling” pathways were down-regulated in PA HB. Dysregulation of notch signalling–associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (<i>NANOG</i>, <i>GATA2</i>, <i>KRT8</i>, <i>LGMN</i>, and <i>DPP4</i>) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.</p></div

Porcine induced pluripotent stem cells produce chimeric offspring

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2-to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology

Primer sequences used in Real-time PCR verification.

<p>Primer sequences used in Real-time PCR verification.</p

QPCR verification result.

<p>QPCR verification result of 14 selected genes. The mRNA expression levels of these genes were normalized with the external control gene (Xeno), and were calculated with 2^−ΔΔCt relative quantification. Bar charts showing the relative expression levels of PSEN2, ANXA8, HES1, JAG1, HEY2, NCSTN, KRT18, KRT8, GATA2, NANOG, SLC36A2, KCTD3, DPP4, and LGMN genes in IVV XB, IVV HB, PA XB, PA HB, and CT HB (KCTD3, SLC36A2, and LGMN genes were not tested in CT HB). The relative expression levels of in each sample were standardized with their expression Error bars shows the standard error (*: P < 0.05). Dashed lines indicate 1.0 expression level. ND: not detected. NT: not tested.</p