ABSTRACT
The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P<0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P<0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass:total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P<0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P<0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.
Subject(s)
Cell Aggregation/physiology , Cloning, Organism/veterinary , Embryo, Mammalian/embryology , Swine, Miniature/embryology , Analysis of Variance , Animals , Apoptosis/physiology , Blastocyst Inner Cell Mass/cytology , CDX2 Transcription Factor , Cloning, Organism/methods , DNA Primers/genetics , Female , Gene Expression Profiling , Homeodomain Proteins/metabolism , Octamer Transcription Factor-3/metabolism , Pregnancy , Pregnancy Outcome , Real-Time Polymerase Chain Reaction , Swine , Trans-Activators/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Matching donor and recipient human leucocyte antigen (HLA-II) could conquer cell-mediated rejection following transplantation. Transgenic pigs carrying HLA genes that "humanize" porcine organs, tissues, and cells were successfully generated. This study further clarifies the effect of HLA-DR transgenes on lymphocyte protein expression, via a proteomic approach. Lymphocytes were isolated from two HLA-DR transgenic pigs and three nontransgenic littermates on 157 d after birth. Soluble protein of 1x10(7) cells was separated using 2-DE. In total, 301 colloidal CBB-stained protein spots detected on all five 2-D gels were quantified. Thirty-three proteins were differentially expressed by a factor of 1.5. These proteins were subsequently identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS. These proteins were sorted into the following categories: chaperones, T-lymphocyte function, DNA/RNA processing, cytoskeleton-associated proteins, signal transduction, enzymes, and unknown. Previous studies have suggested that some of the identified proteins are associated with lymphocyte activation/proliferation. The identities of the unidentified spots and the systematic effect of these up- and down-regulated proteins on T-cell function in HLA-DR transgenic pigs require further exploration.
Subject(s)
HLA-DR Antigens/genetics , Lymphocytes/immunology , Proteome/analysis , Proteomics/methods , Animals , Animals, Genetically Modified , Electrophoresis, Gel, Two-Dimensional , Female , HLA-DR Antigens/physiology , Immunohistochemistry , Isoelectric Point , Male , Molecular Weight , Peptide Mapping , Proteome/classification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Swine , TransgenesABSTRACT
The purpose of this study was to investigate the relationship between swine health status and the concentration of the serum acute phase proteins, haptoglobin (HP), and C-reactive protein (CRP). A total of 378 clinically healthy pigs from farms A and B, plus 20 pigs culled from farm A due to poor growth, were used in this experiment. Each pig was examined and blood samples were collected during slaughter. The HP concentration was measured by using an HP-hemoglobin binding assay. The CRP concentration was measured by using a CRP enzyme immunoassay. Gross and histopathological lesions were examined and recorded at slaughter. Representative samples were then collected in order to isolate pathogens. Swine enzootic pneumonia, found in 47.7% of the pigs, was the most common lesion. Other lesions included pleuropneumonia (32.7%), suppurative pneumonia (10.3%), fibrinous pericardititis (4.3%), Ascaris migration in the liver (33.9%), and intestinal serositis (3.0%). On farm A, the percentage of pigs with 1 or more lesions was 88.2%. For culled pigs from farm A, the mean serum concentrations of HP and CRP were 2.23 +/- 0.14 mg/mL and 252.93 +/- 11.62 microg/mL, which were significantly higher than concentrations in clinically normal pigs (1.42 +/- 0.02 mg/mL and 84.88 +/- 2.61 microg/mL, respectively, P < 0.01). Moreover, among clinically normal farm A pigs, the mean HP concentration in pigs with lesions (1.43 +/- 0.02 mg/mL) was significantly higher than in pigs without lesions (1.32 +/- 0.07 mg/mL) (P < 0.05). However, the mean serum CRP concentrations in these animals were not significantly different. On farm B, the percentage of pigs with one or more lesions was 50.0%. Interestingly, the mean serum HP concentration in clinically normal pigs with lesions was significantly lower in farm B pigs (1.23 +/- 0.07 mg/mL) than in the farm A pigs (1.43 +/- 0.02 mg/mL; P < 0.01). However, serum CRP concentrations in farm A and B pigs were not significantly different. Serum HP concentration, which is a better indicator of inflammatory reactions in pig herds than serum CRP concentration, provides an important marker for swine health status.
