Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids and triacylglycerols and the accumulation of hepatic lipids by increasing mRNA expression related to lipid metabolism, and that excess E(2) in the blood leads to activation of E(2) catabolic metabolism (cytochrome P450 3A37)-related mRNA expression.

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

Transgenic chickens, ubiquitously expressing a human protein, could be a very useful model system for studying the role of human proteins in embryonic development as well as for efficiently producing pharmaceutical drugs as bioreactors. Human parathormone (hPTH) secreted from parathyroid glands plays a significant role in calcium homeostasis and is an important therapeutic agent for the treatment of osteoporosis in humans. Here, by using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein, we generated transgenic chickens expressing hPTH under the control of a ubiquitous Rous sarcoma virus promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage. After 21 d of incubation, 42 chicks hatched from 473 retrovirus-injected eggs. All 42 living chicks were found to express the vector-encoded hPTH gene in diverse organs, as revealed by PCR and reverse transcription-PCR analysis by using primer pairs specific for hPTH. Four days after hatching, 6 chicks died and 14 chicks showed phenotypic deformities. At 18 wk of age, only 3 G(0) chickens survived. They also released the hPTH hormone in their blood and transmitted the hPTH gene to G(1) embryos. However, although the embryos were alive at d 18 of incubation, none hatched. An electrochemiluminescence immunoassay further showed that the hPTH expression level was markedly elevated in mammalian cells infected by the retrovirus vector. Thus, we demonstrated that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, not only could be an efficient bioreactor for the production of pharmaceutical drugs, but also could be useful for studies on the role of human proteins in embryonic development. To our knowledge, this is the first report on the production of a human protein (hPTH) in transgenic chickens under the control of a ubiquitous promoter by using a replication-defective Moloney murine leukemia virus-based retrovirus vector system.

Flavonoid has anti-oxidant properties and has been shown to protect cells against oxygen radical damage. This study therefore, evaluated the effect of flavonoid on the in vitro development ability of porcine embryos produced by parthenogenesis (PA) or somatic cell nuclear transfer (SCNT). Porcine embryos were produced from abattoir-derived prepubertal oocytes either by PA or SCNT of fetal fibroblast into enucleated oocytes essentially as we described earlier (Gupta et al. 2008 Mol. Reprod. Dev. 75, 588–597). One-cell embryos were subsequently cultured in NCSU23 + 0.4% polyvinyl alcohol supplemented with or without 10 μm flavanoid for 7 days at 39°C in a humidified atmosphere of 5% CO2 in air. Results showed that the presence of flavonoid in the culture medium increased (v. controls) the rate of blastocyst in both PA (31.7 ± 4.0 v. 20.4 ± 2.0%) and SCNT (20.6 ± 2.5 v. 12.2 ± 2.9%) groups, respectively (P < 0.05). These blastocysts also had higher ability to hatch (PA: 53.7 ± 3.6 v. 34.0 ± 2.4%; SCNT: 70.9 ± 2.5 v. 45.7 ± 2.9%) and contained higher cell number (PA: 38.9 ± 2.0 v. 31.3 ± 2.1; SCNT: 37.5 ± 2.0 v. 29.7 ± 2.5) than those of control groups (P < 0.05). Western blot analysis of parthenogenetic blastocysts showed that, flavonoid also reduced the expression of caspase-3 and p38 mitogen-activated protein kinase (MAPK) proteins by 3.1 ± 0.1 and 7.7 ± 0.2 fold, respectively while expression of ERK1/2 protein was increased by 4.7 ± 2.3 fold. The 2′,7′-dichlorofluorescene fluorescence staining of embryos further revealed that the activity of reactive oxygen species (ROS) was significantly reduced in flavonoid treated embryos by 2 fold(P < 0.05). These data therefore, suggest that flavonoid may improve the in vitro development rate and quality of porcine embryos by reducing ROS activity and change in protein expression.

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