Identification of protein carbonyls in serum of the fetal and neonatal pig

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITCavidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be ∼600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum

Cross-cutting concepts to transform agricultural research

Agriculture is an important link to many issues that challenge society today, including adaptation to and mitigation of climate change, food security, and communicable and non-communicable diseases in animals and humans. Transformation of agriculture and food systems has become a priority for a range of federal agencies and global organizations. It is imperative that food and agricultural researchers effectively harness the global convergence of priorities to overcome research “silos” through deep and sustained systemic change. Herein, we identify intersections in federal and global initiatives encompassing climate adaptation and mitigation; human health and nutrition; animal health and welfare; food safety and security; and equity and inclusion. Many agencies and organizations share these priorities, but efforts to address them remain uncoordinated and opportunities for collaboration untapped. Based on the interconnectedness of the identified priority areas, we present a research framework to catalyze agricultural transformation, beginning with the research enterprise. We propose that transformation in agricultural research should incorporate (1) innovation, (2) integration, (3) implementation, and (4) evaluation. This framework provides approaches for food and agricultural research to contribute to sustainable, flexible, and coordinated transformation in the agricultural sector

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome

Potential for selection of beneficial traits in swine with site-specific nucleases

Agricultural biotechnology; DNA editing; genome engineering; targetable nucleases; TALEN; CRISPR/cas9; DNA-targeting; mutagenesis; regulation; modern breeding technologies

Isolation and characterization of porcine visceral endoderm cell lines derived from in vivo 11-day blastocysts

Two porcine cell lines of yolk-sac visceral endoderm, designated as PE-1 and PE-2, were derived from in vivo 11-d porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of the cell lines was done on STO feeder cells. The PE-1 and PE-2 cells morphologically resembled visceral endoderm previously cultured from in vivo-derived ovine and equine blastocysts and from in vitro-derived bovine blastocysts. Analysis of the PE-1- and PE-2-conditioned medium by 2D-gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flightmass spectrometry demonstrated that they produced serum proteins. Reverse transcriptase polymerase chain reaction analysis showed that the cells expressed several genes typical for yolk-sac endoderm differentiation and function including GATA-6, DAB-2, REX-1, HNF-1, transthyretin, alpha-fetoprotein, and albumin. Unlike a porcine liver cell line, the PE-1 and PE-2 cell lines had relatively low inducible P-450 content and EROD activity, and, while they cleared ammonia from the cell culture medium, they did not produce urea. Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions and by lateral desmosomes. Rough endoplasmic reticulum was prominent within the cells. Immunocytochemistry indicated that the PE-1 cells expressed cytokeratin 18 and had robust microtubule networks similar to those observed in in vivo porcine yolk-sac endoderm. Metaphase spreads prepared at passage 26 of the PE-1 cell line indicated a diploid porcine karyotype of 38 chromosomes. The cells have been grown for over 1 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The cell lines will be of interest as an in vitro model of the porcine preimplantationy olk-sac tissue

Identification of protein carbonyls in serum of the fetal and neonatal pig

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITCavidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be ∼600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum

ISOLATION AND CHARACTERIZATION OF A BOVINE VISCERAL ENDODERM CELL LINE DERIVED FROM A PARTHENOGENETIC BLASTOCYST

A cell line, BPE-1, was derived from a parthenogenetic 8-d in vitro-produced bovine blastocyst that produced a cell outgrowth on STO feeder cells. The BPE-1 cells resembled visceral endoderm previously cultured from blastocysts produced by in vitro fertilization (IVF). Analysis of the BPE-1 cells demonstrated that they produced serum proteins and were negative for interferon-tau production (a marker of trophectoderm). Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions in conjunction with desmosomes. Rough endoplasmic reticulum was prominent within the cells as were lipid vacuoles. Immunocytochemistry indicated the BPE-1 cells had robust microtubule networks. These cells have been grown for over 2 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The BPE-1 cell line presumably arose from embryonic cells that became diploid soon after parthenogenetic activation and development of the early embryo. However, metaphase spreads prepared at passage 41 indicated that the cell population had a hypodiploid (2n = 60) unimodal chromosome content with a mode of 53 and a median and mean of 52. The cell line will be of interest for functional comparisons with bovine endoderm cell lines derived from IVF and nuclear transfer embryos

Metabolomic analysis of longissimus from underperforming piglets relative to piglets with normal preweaning growth

Abstract Background Recent increases in intra-litter variability in weaning weight have raised swine production costs. A contributor to this variability is the normal birth weight pig that grows at a slower rate than littermates of similar birth weight. The goal of this study was to interrogate biochemical profiles manifested in skeletal muscle originating from slow growing (SG) and faster growing littermates (control), with the aim of identifying differences in metabolic pathway utilization between skeletal muscle of the SG pig relative to its littermates. Samples of longissimus muscle from littermate pairs of pigs were collected at 21 d of age for metabolomic analysis (Metabolon, Inc., Durham, NC). Results Birth weights did not differ between littermate pairs of SG and Control pigs (P > 0.05). Weaning weights differed by 1.51 ± 0.19 kg (P < 0.001). Random forest (RF) analysis was effective at segregating the metabolome of muscle samples by growth rate, resulting in a predictive accuracy of 81% versus random segregation (50%). Decreases in sugars in the pentose phosphate pathway (PPP) in the longissimus of SG pigs were detected (P < 0.05). Decreases were also apparent in glycolytic intermediates (glycerol-3-phosphate and lactate) and key glycolysis-derived intermediates (glucose-6-phosphate and fructose-6-phosphate; P < 0.05). SG pigs had increased levels of phospholipids, lysolipids, diacylglycerols, and sphingolipids (P < 0.05). Pathway analysis identified a cluster of molecules associated with muscle and collagen/extracellular matrix breakdown that are increased in the SG pig (glutamate, 3-methylhistidine and hydroxylated proline moieties; P < 0.05). Nicotinate metabolism was altered in SG pigs, resulting in a 78% decrease in the nicotinamide adenine dinucleotide pool (P < 0.05). Conclusions These metabolomic data provide the first evidence for biochemical mechanisms that should be investigated to determine if they have a potential role in the slow growth in some normal birth weight piglets that contribute to increased intra-litter variability in weaning weights and provides essential information and potential targets for the development of nutritional intervention strategies