Alpha-lipoic acid improves the reproduction performance of breeder hens during the late egg-laying period.

Alpha-lipoic acid (ALA), a multifunctional antioxidant, can promote fatty acid mobilization, energy expenditure and scavenge free radicals. The effects of dietary ALA on the reproductive performance of breeder hens were investigated in the current study. In the 5-week experiment, 180 54-week-old Qiling breeder hens were randomly divided into three treatments with five replicates and supplemented with three levels of ALA (0, 300 and 600 mg/kg) in the basic corn-soya bean meal diets. 600 mg/kg ALA treatment group (HLA) significantly improved the eggshell thickness and strength (p < .05). ALA-treated groups improved egg-laying rate compared with the CON group, but with no statistically significant difference (p > .05). The levels of HDL-C, ALB and estradiol (E2) of the serum in the HLA group were elevated compared with the CON group (p < .05). In addition, ALA (600 mg/kg) treatment exhibited a reduced level of serum AST and TG (p < .05). Dietary ALA increased the activity of hepatic lipase in liver (p < .05). Supplemental 600 mg/kg ALA also improved the SOD activity and total antioxidant capacity level, along with a decreased MDA in ovarian tissue (p < .05). Furthermore, the mRNA expressions of ESR1, ESR2, VTG2 and ApoB in the liver and FSHR in follicles were upregulated in the HLA group (p < .05). In conclusion, dietary supplementation with 600 mg/kg ALA during the late egg-laying period could improve lipid metabolism and reproductive performance of breeder hens.

Dietary genistein supplementation protects against lipopolysaccharide-induced intestinal injury through altering transcriptomic profile.

Genistein is abundant in the corn-soybean meal feed. Little information is available about the effect of dietary genistein on the intestinal transcriptome of chicks, especially when suffering from intestinal injury. In this study, 180 one-day-old male ROSS 308 broiler chickens were randomly allocated to 3 groups, with 4 replicates (cages) of 15 birds each. The treatments were as follows: chicks received a basal diet (CON), a basal diet and underwent lipopolysaccharide-challenge (LPS), or a basal diet supplemented with 40 mg/kg genistein and underwent LPS-challenge (GEN). LPS injection induced intestinal injury and inflammatory reactions in the chicks. Transcriptomic analysis identified 7,131 differently expressed genes (3,281 upregulated and 3,851 downregulated) in the GEN group compared with the LPS group (P adjusted value < 0.05, |fold change| > 1.5), which revealed that dietary genistein exposure altered the gene expression profile and signaling pathways in the ileum of LPS-treated chicks. Furthermore, dietary genistein improved intestinal morphology, mucosal immune function, tight junction, antioxidant activity, apoptotic process, and growth performance, which were adversely damaged by LPS injection. Therefore, adding genistein into the diet of chicks can alter RNA expression profile and ameliorate intestinal injury in LPS-challenged chicks, thereby improving the growth performance of chicks with intestinal injury.

RNA Expression Profile and Alternative Splicing Signatures of Genistein-Treated Breeder Hens Revealed by Hepatic Transcriptomic Analysis.

Little information has been available about the influence of dietary genistein (GEN) on hepatic transcriptome of laying broiler breeder (LBB) hens. The study is aimed at broadening the understanding of RNA expression profiles and alternative splicing (AS) signatures of GEN-treated breeder hens and thereby improving laying performance and immune function of hens during the late egg-laying period. 720 LBB hens were randomly allocated into three groups with supplemental dietary GEN doses (0, 40 mg/kg, and 400 mg/kg). Each treatment has 8 replicates of 30 birds. Dietary GEN enhanced the antioxidative capability of livers, along with the increased activities of glutathione peroxidase and catalase. Furthermore, it improved lipid metabolic status and apoptotic process in the liver of hens. 40 mg/kg dietary GEN had the better effects on improving immune function and laying performance. However, transcriptome data indicated that 400 mg/kg dietary GEN did negative regulation of hormone biosynthetic process. Also, it upregulated the expressions of EDA2R and CYR61 by the Cis regulation of neighbouring genes (lncRNA_XLOC_018890 and XLOC_024242), which might activate NF-κB and immune-related signaling pathway. Furthermore, dietary GEN induced AS events in the liver, which also enriched into immune and metabolic process. Therefore, the application of 40 mg/kg GEN in the diet of breeder hens during the late egg-laying period can improve lipid metabolism and immune function. We need to pay attention to the side-effects of high-dose GEN on the immune function.

Effects of High-Dose Genistein on the Hypothalamic RNA Profile and Intestinal Health of Female Chicks.

Genistein is abundant in animal feed. In this study, the side effects of high-dose genistein on intestinal health and hypothalamic RNA profile were evaluated. Chicks exposed to high-dose genistein by intraperitoneal injection (416 ± 21, 34.5 ± 2.5) and feed supplementation (308 ± 19, 27.2 ± 2.1) both showed a reduced body weight gain and feed intake in comparison with the control group (261 ± 16, 22.7 ± 1.6, P < 0.01). In comparison with the control (22.4 ± 0.5, 33.3 ± 2.4), serum levels of albumin and total protein were decreased after high-dose genistein injection (21.6 ± 0.5, 31.8 ± 1.6) and diet supplementation (20.6 ± 0.9, 29.9 ± 2.5, P < 0.001). Interestingly, the genistein diet presented the chick hypothalamus with downregulated expression of bitter receptors (TAS1R3, P < 0.05). Meanwhile, it upregulated the expressions of TAS2R1 (P < 0.05) and downstream genes (PLCB2 and IP3R3) in the ileum (P < 0.05). Accordingly, high-dose dietary genistein reduced villus height and the abundance of Lactobacillus, along with the increased abundance of pathogenic bacteria in the ileum (P < 0.05). Furthermore, transcriptomic analysis identified 348 differently expressed genes (168 upregulated and 224 downregulated) in the high-dose dietary genistein treated group in comparison with the control (P < 0.05, |log2FoldChange| > 0.585). Therefore, high-dose dietary genistein altered the hypothalamic RNA profile and signal processing. Cluster analysis further revealed that high-dose dietary genistein significantly influenced apoptosis, the immune process, and the whole synthesis of steroid hormones in the hypothalamus (P < 0.05). In conclusion, high-dose dietary genistein altered the hypothalamic RNA profile and intestinal health of female chicks.

Dietary Stevioside Supplementation Alleviates Lipopolysaccharide-Induced Intestinal Mucosal Damage through Anti-Inflammatory and Antioxidant Effects in Broiler Chickens.

The study was conducted to investigate the effects of dietary stevioside (STE) supplementation on the lipopolysaccharide (LPS)-induced intestinal mucosal damage of broiler chickens. A total of 192 one-day-old male Ross 308 broiler chicks were randomly divided into four treatments: (1) basal diet (CON); (2) basal diet supplemented with 250 mg/kg stevioside (STE); (3) basal diet + LPS-challenge (LPS); (4) basal diet supplemented with 250 mg/kg stevioside + LPS-challenge (LPS + STE). LPS-challenged groups received an intraperitoneal injection of LPS at 17, 19 and 21 d, whereas the CON and STE groups received a saline injection. The results showed that dietary STE supplementation normalized LPS-induced changes in protein expression of p-NF-κB and p-IκBα, mRNA expression of inflammatory genes (TLR4, NF-κB, and IFN-γ), tight junction-related genes (CLDN2, OCLN, and ZO-1), and antioxidant genes (Nrf2 and HO-1). LPS-induced decreases in serum diamine oxidase (DAO) level, villus height-to-crypt depth ratio, apoptotic index, and protein expression of proliferating cell nuclear antigen (PCNA) were reversed with dietary STE supplementation. Additionally, STE supplementation ameliorated the redox damage by reducing malondialdehyde (MDA) content and increasing total antioxidant capacity (T-AOC) and antioxidant enzyme activity. In conclusion, dietary stevioside supplementation could alleviate LPS-induced intestinal mucosal damage through anti-inflammatory and antioxidant effects in broiler chickens.

Cysteamine increases expression and activity of H+-K+-ATPase of gastric mucosal cells in weaning piglets.

AIM: To determine the in vivo and in vivo effects of cysteamine (CS) on expression and activity of H(+)-K(+)-ATPase of gastric mucosal cells in weaning piglets. METHODS: Eighteen litters of newborn Xinhuai piglets were employed in the in vivo experiment and allocated to control and treatment groups. From 12 d of age (D12), piglets in control group were fed basal diet, while the treatment group received basal diet supplemented with 120 mg/kg CS. Piglets were weaned on D35 in both groups. Six piglets from each group (n = 6) were slaughtered on D28 (one week before weaning), D35 (weaning), D36.5, D38, D42, and D45 (36 h, 72 h, one week and 10 d after weaning), respectively. Semi-quantitative RT-PCR was performed to determine the levels of H(+)-K(+)-ATPase mRNA in gastric mucosa. H(+)-K(+)-ATPase activity in gastric mucosa homogenate was also determined. Gastric mucosal epithelial cells from piglets through primary cultures were used to further elucidate the effect of CS on expression and activity of H(+)-K(+)-ATPase in vivo. Cells were treated for 20 h with 0.001, 0.01, and 0.1 mg/mL of CS (n = 4), respectively. The mRNA expression of H(+)-K(+)-ATPase and somatostatin (SS) as well as the H(+)-K(+)-ATPase activity were determined. RESULTS: in vivo, both mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of control group exhibited a trend to increase from D28 to D45, reaching a peak on D45, but did not show significant age differences. Furthermore, neither the mRNA expression nor the activity of H(+)-K(+)-ATPase was affected significantly by weaning. CS increased the mRNA expression of H(+)-K(+)-ATPase by 73%, 53%, 30% and 39% on D28 (P = 0.014), D35 (P = 0.017), D42 (P = 0.013) and D45 (P = 0.046), respectively. In accordance with the mRNA expression, H(+)-K(+)-ATPase activities were significantly higher in treatment group than in control group on D35 (P = 0.043) and D45 (P = 0.040). In vivo, CS exhibited a dose-dependent effect on mRNA expression and activity of H+-K+-ATPase. Both H(+)-K(+)-ATPase mRNA expression and activity in gastric mucosal epithelial cells were significantly elevated after 20 h of exposure to the moderate (H(+)-K(+)-ATPase expression: P=0.03; H(+)-K(+)-ATPase activity: P = 0.014) and high concentrations (H(+)-K(+)-ATPase expression: P=0.017; H(+)-K(+)-ATPase activity: P = 0.022) of CS. Significant increases in SS mRNA expression were observed to accompany the elevation of H(+)-K(+)-ATPase expression and activity induced by the moderate (P = 0.024) and high concentrations (P = 0.022) of CS. Low concentration of CS exerted no effects either on expression and activity of H(+)-K(+)-ATPase or on SS mRNA expression in cultured gastric mucosal epithelial cells. CONCLUSION: No significant changes are observed in mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of piglets around weaning from D28 to D45. CS increases expression and activity of gastric H(+)-K(+)-ATPase in vivo and in vivo. SS is involved in mediating the effect of CS on gastric H(+)-K(+)-ATPase expression and activity in weaning piglets.

[The developmental changes of GHR and IGF-1R gene expressions in porcine hypothalamus and pituitary].

GH and IGF-1 may serve as negative feedback factors to regulate GH secretion from pituitary by binding to their respective receptors in hypothalamus and/or pituitary. In order to evaluate the line-specific developmental patterns of negative feedback regulation of GH secretion, Erhualian (EHL) and Large White (LW) pigs with significant difference in growth rate were employed in present study to investigate the developmental changes of GH receptor (GHR) mRNA and type-1 IGF receptor (IGF-1R) mRNA in hypothalamus and pituitary from birth till 180 days of age by relative quantitative RT-PCR. Pigs were sampled at birth, 3 , 20, 30, 90, 120 and 180 days of age respectively. Hypothalamic GHR mRNA was expressed according to an age-dependent manner, being low at birth, then increased steadily till day 120, followed by a decrease (P < 0.05) at the age of 180 days, suggesting that the sensitivity of hypothalamus to the GH negative feedback influence increase steadily during fast-growing period. LW boars expressed higher level of GHR mRNA than EHL boars (P < 0. 05) in hypothalamus. In pituitary, however, the GHR mRNA level was not significantly correlated with the breeds and age. The results suggested that GH might act mainly at the level of hypothalamus to regulate GH secretion. In contrast, the expression of IGF-1R mRNA exhibited line-specific developmental patterns in pituitary but not in hypothalamus. Hypothalamic expression of IGF-1R mRNA was abundant but did not show significant differences between ages, groups or lines. In pituitary, however, the IGF-1R mRNA expression was found to be high at birth both in EHL and LW pigs, subsequently declined till day 20, then followed by a slow rise reaching the second peak at the age of 90 days. At the age of 180 days, the pituitary IGF-1R mRNA level was higher in EHL pigs than that in LW pigs (P < 0.05), but the opposite was true at the age of 30 and 90 days. These results suggest that the site for receiving the feedback signal of IGF-1 is more likely in pituitary rather than in hypothalamus in the pig.

Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs.

The present study was aimed to investigate the developmental patterns of leptin mRNA expression in dorsal subcutaneous adipose tissue and Ob-Rb mRNA expression in hypothalamus in pigs of different breeds and sexes. Erhualian gilts and boars and Large White boars were sampled at birth, 3, 20, 30, 45, 90, 120 and 180 days of age, respectively. Serum concentration of leptin was measured with RIA and single tube semi-quantitative RT-PCR was applied to determine the relative abundances of mRNA expression using 18S rRNA as an internal standard. The results showed that leptin mRNA expression in adipose tissue increased with age and displayed both sex and breed differences. In Erhualian pigs, females expressed higher leptin mRNA compared with males, and Erhualian boars showed higher abundance of leptin mRNA than Large White boars (P < 0.01). Serum leptin levels were in good agreement with adipose leptin mRNA, displaying similar sex and line differences. In contrast, expression of Ob-Rb mRNA in hypothalamus exhibited a distinctive pattern, decreased gradually after birth, and then increased till weaning. After weaning, Ob-Rb gene expression decreased gradually with age but rose gradually again from 120 to 180 days of age in Erhualian pigs. The expression of Ob-Rb mRNA was higher in Large White pigs than that in Erhualian pigs (P< 0.01). The results suggest that the serum leptin level and leptin gene expression in adipose tissue highly correlate with adiposity.

[The developmental patterns of GH-R, IGF-1 and IGF-IR gene expression in adipose tissue of Erhualian and large white pigs].

Growth hormone (GH) is essential for postnatal growth in animal, it regulates numerous cellular functions by direct effect on its receptor (GH-R) in many different tissues. In present experiment single tube semi-quantitative RT-PCR was applied to investigate the developmental patterns of GH-R, IGF-1 and IGF-I R mRNA expression in dorsal subcutaneous adipose tissue of Erhualian and Large White pigs, and 18S internal standards were used as control. Eight Erhualian pigs were sampled at birth, 3, 20, 30, 45, 90, 120, 180 days of age respectively, four Large White boars were sampled at 20, 30, 90, 120, 180 days of age respectively. The results showed that GH-R mRNA levels in adipose tissue were changed with age (P < 0.05). The expression of GH-R mRNA in Erhualian gilts was low at birth but reached a peak at 45 days of age, and kept constant thereafter. The expression of GH-R mRNA in Erhualian boars was high at birth and reached a peak at 45 day, then decreased remarkably. It had no differences between sexes in general. The expression of GH-R mRNA in Large White boars was lower than that in Erhualian boars (P < 0.01). The IGF-1 mRNA expression in adipose tissue was changed with age. The IGF-1 mRNA expression in Erhualian gilts was lower than that in Erhualian boars (P < 0.05), and the IGF-1 mRNA expression in Large White boars was lower than that in Erhualian boars (P < 0.01). The IGF-I R mRNA expression in adipose tissue was changed with aging, but it had no differences between sexes and breeds in general. The results suggest that the GH-R, IGF-1 and IGF-I R mRNA expression in adipose tissue follow specific developmental pattern, and the different patterns of IGF-1 gene expression may contribute to different sediment of adipose tissue between two breeds.

Developmental patterns of GHr and SS mRNA expression in porcine gastric tissue.

AIM: The present study was aimed to investigate the developmental patterns of growth hormone receptor (GHr) and somatostatin (SS) mRNA expression in porcine gastric tissue and its relationship with gastric growth and gastric functional development. METHODS: Erhualian and Large White boars were selected randomly and sampled at birth (D0), 3, 20, 30, 90, 120 and 180 days of age respectively, meanwhile the bodyweight and gastric weight were recorded. The single tube semi-quantitative RT-PCR was applied in this experiment to investigate the developmental patterns of gastric GHr and SS mRNA expression, the correlations between the patterns of mRNA expression and the relative gastric weight (ratio of gastric weight to bodyweight) and the pepsin contents in gastric mucous membrane were analyzed. In order to further elucidate the effect of GH on gastric function, the primary cultures of gastric fundic mucosal cells were treated with 2 ng/ml, 20 ng/ml and 200 ng/ml of rpGH for 18 hrs respectively, and the pepsin contents in culture medium were measured. RESULTS: The expression of GHr mRNA was high at birth, followed by a significant decrease at 3 days of age (D3) in both breeds. In Large White boars, the expression of GHr mRNA reached a peak at D90 and remained a plateau afterward. In Erhualian pigs, however, a slight yet significant increase occurred at D30 reaching a level that was kept constant thereafter. From birth to D30, the expression of GHr mRNA in gastric tissue was higher in Erhualian boars than that in Large White boars, but from D90 to D180, the higher expression of GHr mRNA was found in Large White boars. The gastric GHr mRNA expression was significantly correlated with the relative gastric weight (r=0.541, P<0.05) and pepsin content in gastric mucosa (r=0.625, P<0.05) respectively. The gastric SS mRNA expression was high at birth (Erhualian 1.59, Large White 0.80), but dropped significantly at D3 (Erhualian 0.95, Large White 0.19, P<0.05), a stepwise increase was followed thereafter until a peak at D30 (Erhualian 1.71, Large White 0.95) In general, Erhualian pigs expressed higher levels of SS mRNA in gastric tissue as compared with Large White pigs at the same age (P<0.05). No significant correlations between SS mRNA and relative gastric weight or pepsin content were found. In vitro, 2 ng/ml of rpGH elicited significant increase in pepsin secretion from primary cultures of gastric mucosal cells (P<0.05), and no responses were observed at 20 ng/ml and 200 ng/ml. CONCLUSION: The results suggested that: 1, GHr and SS mRNA in porcine stomach are expressed according to strain specific developmental patterns; 2, GH acts directly at the gastric tissue and regulates the structural and functional growth of stomach.