BackgroundAs an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.ResultsThe mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.ConclusionThere was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

The expression of mRNAs for transforming growth factors (TGF-beta2, myostatin, activin-B, and follistatin), insulin-like growth factors (IGF-I and -II), and fibroblast growth factor (basic, bFGF) was investigated in satellite cells derived from chicken pectoralis major (PM) and biceps femoris (BF) muscles in the stages from initiation of proliferation to fusion. These growth factor gene cDNAs were synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). No myostatin, activin-B, follistatin or bFGF expression was detected in either cell culture at 0 h. TGF-beta2 mRNA level increased at 48 h (P < 0.01) and remained constant through 144 h in both PM and BF satellite cell cultures. The ontogeny of myostatin gene expression with the exception of a sharp increase in BF culture at 72 h (P < 0.01), was nearly identical in both cell cultures. Activin-B mRNA level in PM satellite cells was higher than that in BF satellite cells at 72 h and 120 h (P < 0.01). Follistatin mRNA in PM satellite cells was higher than that in BF satellite cells at 24, 96, and 120 h culture (P < 0.01). No IGF-I gene expression was detected in cell cultures at any time point. IGF-II gene expression in BF satellite cells declined at 96 h (P < 0.01) and remained reduced until 144 h. bFGF mRNA in both satellite cell cultures increased at 24 h (P < 0.05) and remained at this level in BF satellite cells through 144 h.

Two experiments assessed the efficacy of in ovo administration of insulin-like growth factor-I (IGF-I) to enhance skeletal muscle development and improve feed efficiency of broilers. Hatching eggs were divided into three groups: uninjected control, vehicle-injected control, and recombinant human (rh) IGF-I (100 ng per embryo). Eggs in Experiment 1 were injected on Day 1, 4, or one of Day 7 through 18 of incubation. Growth rates for Days 1 and 4 resulted in the greatest response to treatment (P < 0.01, P < 0.06 respectively). Based on these results, Experiment 2 focused on Days 1 to 4 of incubation. Results from Experiment 2 showed that there was no significant difference in hatchability among control and rh IGF-I treatment groups. Injection on Day 3 resulted in the greatest response for increased live (P < 0.035) and leg (P < 0.02) weights in both sexes. Feed efficiencies of all rh IGF-I groups were significantly (P < 0.01) improved for the first 3 wk. In ovo administration of rh IGF-I on Day 3 increased feed efficiency (6.65%; P < 0.009) in pens of mixed-sex broilers. In addition, live weights (12.3%; P < 0.002), leg weights (11.7%; P < 0.01), breast weights (9.9%; P < 0.04), and heart weights (11.4%; P < 0.02) were increased in males. These results demonstrate that in ovo administration of rh IGF-I alters feed efficiency, growth, and tissue development. This finding lends itself to significant improvements in broiler production efficiency and profitability.

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Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to demonstrate whether a correlation exists between insulin-like growth factors (IGFs)-positive regulators of growth-and myostatin, a negative regulator of muscle growth. IGF-I, -II, and IGF-receptor-1 (IGF-R1) mRNA and IGF-II protein expressions were determined in control and myostatin knockout mice tissues. IGF-I gene expressions were similar between control and knockout mice tissues, whereas IGF-II mRNA levels were significantly higher in myostatin knockout mice kidney and soleus muscles than those of control mice (P <.01). IGF-R1 mRNA levels from control mice heart (P <.05) and kidney (P <.01) were significantly higher than in myostatin knockout mice, whereas levels were lower in pectoralis muscle of control mice than knockout mice (P <.01). The strongly IGF-II-positive cells in soleus muscle were more common in myostatin knockout mice and were seen in a few foci in control mice. IGF-II immunoreactivity in both control and myostatin knockout mice kidneys was localized to the epithelium of renal tubules and collecting ducts. Reciprocal changes in the expression of myostatin and IGF-II and IGF-R1 may underlie normal growth of skeletal muscle and other organs in mammals, and the changes in these tissues associated with disease.