Melatonin protects porcine oocyte from copper exposure potentially by reducing oxidative stress potentially through the Nrf2 pathway.

Copper is widely used as a feeding additive to promote livestock growth. However, excessive copper can be excreted with feces, causing heavy metal pollution and aggravating environmental problems. At the same time, studies have found that excess copper can cause damage to reproductive function and reduce gamete quality. Here, we explored the effects of adding different concentrations of copper to the culture medium on porcine oocytes. First polar body extrusion rate, embryo development, and intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) ΔΨm, adenosine triphosphate(ATP) content, and acetylation of lysine 9 on histone H3 protein subunit (H3K9ac) were assessed. Results demonstrated that Cu exposure causes abnormalities in mitochondrial function and epigenetic modification, resulting in increased oxidative stress and levels of ROS, ultimately leading to a decreased porcine oocyte quality. In addition, we found melatonin can protect porcine oocytes from those damages. Notably, Nrf2 protein expression was significantly increased by copper exposure, meanwhile, Nrf2 signaling pathway inhibitor ML385 significantly attenuated the protective role of melatonin on oxidative stress induced by copper exposure. In summary, our study demonstrates that copper activates the Nrf2 pathway and impairs oocyte maturation by inducing oxidative stress, leading to poor quality of porcine oocytes, and the changes can be reversed by melatonin.

Arginine Regulates Zygotic Genome Activation in Porcine Embryos Under Nutrition Restriction.

Arginine has a positive effect on pre-implantation development in pigs. However, the exact mechanism by which arginine promotes embryonic development is undefined. Here, single-cell RNA sequencing technology was applied to porcine in vivo pre-implantation embryos from the zygote to morula stage, it was found that that the expression of arginine metabolism-related genes clearly changed from the 2-cell stage to the 4-cell stage, when zygotic genome activation (ZGA) occurs in porcine embryos. Further analysis showed that arginine metabolism-related genes are significantly correlated with key ZGA genes. To determine the function of arginine in porcine embryos during ZGA, the in vitro fertilization embryos were cultured in PZM-3 medium (0.12 mM arginine, Control group), a modified PZM-3 medium (0 mM arginine, Block group) and a modified PZM-3 medium supplemented with arginine (0.12 mM arginine, Block + Arg group). The results showed that the 4-cell arrest rate was significantly increased in the Block group compared to the Control group (P < 0.05). The 4-cell arrest rate in the Block + Arg group was significantly decreased than that in the Block group (P < 0.05). Meanwhile, the expression of ZGA marker genes and SIRT1 protein in 4-cell embryos was significantly decreased in the Block group compared to the Control group, and their expression was significantly increased in the Block + Arg group. In addition, we observed that the glutathione (GSH), ATP levels, and lipid droplet contents were significantly increased, and the reactive oxygen species (ROS) level was decreased in the Block + Arg group compared to the Block group. Compared with Control group, spermine content in culture medium and the mRNA expression of ornithine decarboxylase1 (ODC1) of embryos in the Block group were significantly decreased (P < 0.05), and those in the Block + Arg group were significantly increased compared with the Block group (P < 0.05). Moreover, when difluoromethylornithine (an inhibitor of ODC1) was added to the modified PZM-3 medium supplemented with arginine, the effect of arginine on ZGA was inhibited. In summary, our findings demonstrated that arginine may regulate ZGA under nutrition restriction in porcine embryos by promoting polyamine synthesis.

Effects of pyruvate on early embryonic development and zygotic genome activation in pigs.

Pyruvate is an important energy substance during early embryonic development of mammals. However, the underlying mechanisms of pyruvate during early embryonic development in pigs and its role in zygotic genome activation (ZGA) are not fully understood. Here, based on a previous RNA-seq dataset of porcine early embryos, we found that pyruvate metabolism-related genes started to be expressed at the 4-cell stage and that pyruvate metabolism-related genes were correlated with porcine ZGA marker genes. To determine the function of pyruvate in porcine embryos, in vitro fertilization (IVF) embryos were cultured in PZM-3 medium (control group); modified PZM-3 medium that only contains pyruvate and lactate plus salts (+P group); or modified PZM-3 medium lacking pyruvate (-P group). The 4-cell arrest rate at 72 h was significantly increased in the -P group compared to the +P group (P < 0.05). In addition, we observed that the reactive oxygen species (ROS) level was significantly increased and that the adenosine triphosphate (ATP) level was significantly (P < 0.05) decreased in the -P group compared to the +P group. Moreover, the expression of ZGA marker genes and SIRT1 protein in embryos was significantly decreased in the -P group compared to the +P group (P < 0.05). Furthermore, the acetylation level of H3K9 was significantly decreased (P < 0.05) and the methylation level of H3K9 was significantly increased (P < 0.05) in the -P group compared to the +P group. In summary, our findings demonstrate that pyruvate affects early embryonic development in pigs by promoting ZGA and reducing oxidative stress levels.

Quercetin Reduces Inflammation and Protects Gut Microbiota in Broilers.

The aim of this study was to investigate the effects of quercetin on inflammatory response and intestinal microflora in broiler chicken jejuna. A total of 120 broiler chickens were allocated into 3 groups: saline-challenged broilers fed a basal diet (CTR group), lipopolysaccharide (LPS)-challenged broilers fed a basal diet (L group) and LPS-challenged broilers fed a basal diet supplemented with 200 mg/kg quercetin (LQ group). Our results showed that LPS significantly increased expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, interferon (IFN)-γ, toll-like receptor (TLR)-4, Bax, Caspase-3 and diamine oxidase activity (DAO), and decreased expression of zona occludens-1 (ZO-1), Occludin and Bcl-2 in the jejunum, while dietary quercetin prevented the adverse effects of LPS injection. LPS injection significantly decreased the number of Actinobacteria, Armatimonadetes and Fibrobacteriae at the phylum level when compared to the CTR group. Additionally, at genus level, compared with the CTR group, the abundance of Halomonas, Micromonospora, Nitriliruptor, Peptococcus, Rubellimicrobium, Rubrobacter and Slaclda in L group was significantly decreased, while dietary quercetin restored the numbers of these bacteria. In conclusion, our results demonstrated that dietary quercetin could alleviate inflammatory responses of broiler chickens accompanied by modulating jejunum microflora.

Gonadotropin-inhibiting hormone promotes apoptosis of bovine ovary granulosa cells.

Gonadotropin-inhibiting hormone (GnIH) inhibits the synthesis and release of gonadotropin by binding to its receptor. GnIH is involved in animal reproductive regulation, especially ovary function. It can regulate the proliferation, apoptosis and hormone secretion of follicular cells. However, the role and molecular mechanism of GnIH in bovine granulosa cell (bGC) apoptosis is unclear. Here, the effects of GnIH on proliferation, apoptosis, and mitochondrial function of bGCs were detected. A 10-6 mol/mL concentration of GnIH inhibited bGC proliferation, promoted GC apoptosis, and damaged mitochondrial function. Additionally, GnIH significantly decreased the phosphorylation level of p38 (P < 0.01). To explore the role of the p38 signaling pathway in the process of GnIH-induced apoptosis in bGCs, an activator of p38 (U46619) was used to pretreat bGCs. U46619 pretreatment significantly alleviated GnIH damage to bGCs, including proliferation, apoptosis, and mitochondrial function. In conclusion, these results demonstrated that GnIH inhibited proliferation and promoted apoptosis of bGCs via the p38 signaling pathway.

Correlations between circulating leptin concentrations and growth performance, carcass traits, and meat quality indexes in finishing Simmental × Luxi bulls fed high-concentrate diets.

Circulating leptin concentrations could potentially be used as a predictor of production traits in cattle. This study aimed to clarify the correlations between circulating leptin concentrations and growth performance, carcass traits, and meat quality indexes in finishing bulls fed high-concentrate diets (concentrate-to-forage ratio 70:30). Fifty-seven Simmental × Luxi F1 crossbred bulls were used for 112-day finishing experiment. Circulating leptin concentrations and relevant indexes of growth performance, and carcass traits and meat quality were measured during or after finishing trail. The results indicated that the leptin concentrations tended to be negatively correlated with dry matter intake (DMI) (r = -.233, p = .081), and were positively correlated with 12th-rib fat thickness (r = .330, p = .012), marbling score (r = .336, p = .011), and intramuscular fat content (r = .368, p = .021). Moreover, the leptin concentrations were negatively correlated with cholesterol content (r = -.339, p = .037) and were not correlated with sensory indexes including tenderness, juiciness, and like flavor (p > .05). In conclusion, circulating leptin concentrations may potentially be used as a predictor of carcass traits related to content of fat and beef quality traits related to content of cholesterol in finishing bulls fed high-concentrate diets.

Quercetin Protects Against Lipopolysaccharide-Induced Intestinal Oxidative Stress in Broiler Chickens through Activation of Nrf2 Pathway.

We investigated the potential ability of quercetin to protect against lipopolysaccharide (LPS)-induced intestinal oxidative stress in broiler chickens and the potential role of the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway. One-day-old broiler chickens (n = 240) were randomized into four groups: saline-challenged broiler chickens fed a basal diet (Con), LPS-challenged broiler chickens on a basal diet (LPS), and LPS-treated broiler chickens on a basal diet containing either 200 or 500 mg/kg of quercetin (Que200+LPS or Que500+LPS). Quercetin (200 mg/kg) significantly alleviated LPS-induced decreased duodenal, jejunal, and illeal villus height and increased the crypt depth in these regions. Quercetin significantly inhibited LPS-induced jejunal oxidative stress, including downregulated reactive oxygen species (ROS), malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, and it upregulated superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. Quercetin relieved LPS-induced jejunal mitochondria damage and upregulated mitochondrial DNA copy number-related gene expression, including cytochrome c oxidase subunit 1 (COX1), ATP synthase F0 subunit 6 (ATP6), and NADH dehydrogenase subunit 1 (ND1). Quercetin attenuated the LPS-induced inhibition of Nrf2 activation, translocation, and downstream gene expression, including heme oxygenase-1 (HO-1), NAD (P) H dehydrogenase quinone 1 (NQO1), and manganese superoxide dismutase (SOD2). Additionally, quercetin attenuated the LPS-inhibition of c-Jun N-terminal kinase (JNK), Extracellular Regulated protein Kinases (ERK), and p38MAPK (p38) phosphorylation in the MAPK pathway. Thus, quercetin attenuated LPS-induced oxidative stress in the intestines of broiler chickens via the MAPK/Nrf2 signaling pathway.

Photoperiodic effect on the testicular transcriptome in broiler roosters.

Information about the effects of photoperiod on the testicular transcriptome of broiler roosters is limited. The aim of the present study was to explore the effect of different photoperiodic regimes on gene expression in the testes of broiler breeder roosters. One hundred and twenty Arbor Acres broiler breeder roosters aged 20 weeks were assigned to one of three groups (n = 40) and subjected to different photoperiodic regimes: control (CTR; 12.5 L:11.5 D), short day (SD; 8 L:16 D) and long day (LD; 16 L:8 D). After 4 weeks, the testes of 10 randomly selected birds from each group were dissected, sliced and haematoxylin-eosin stained. The testicular transcriptome of roosters from the SD and LD groups was determined by RNA sequencing (RNA-Seq), and the results were confirmed using quantitative real-time PCR. The seminiferous tubule area and sperm count increased significantly with the prolongation of photoperiod (p < .01). Additionally, the RNA-Seq results indicated that 387 genes were upregulated and 1,052 genes were downregulated in the LD group compared with those in the SD group. Several crucial genes involved in rooster testicular development and reproduction were also screened, including heat shock proteins 90, extracellular regulated protein kinases 1, phosphatidylinositol 3-kinase, adenosine 5'-monophosphate -activated protein kinase, BCL-6 and Smad3. The differentially expressed genes were enriched in the mammalian targets of rapamycin (mTOR), forkhead box (FoxO), transforming growth factor beta (TGF-ß) and insulin signalling pathway. In conclusion, a 16 hr photoperiod for 4 weeks increased the seminiferous tubule duct area and promoted spermatogenesis in the rooster's testicles, and the mTOR, FoxO, TGF-ß and insulin signalling pathways may be involved.

Advances in the mechanism of high copper diets in restraining pigs growth.

High copper feed has been widely used as an inexpensive and highly effective feed additive to promote growth performance of pigs. However, long-term feeding of high copper feed may reduce the growth-promoting effects of copper, time-dependent accumulation of copper in animal tissues and organs, and copper toxicity thereby reducing the growth performance of pigs. Due to the widespread effects of high copper supplementation in animals' diets, the benefits and drawbacks of high copper feeding in pigs have been reported in several studies. Meanwhile, few of these studies have systematically described the mechanism by which high copper diets restrain pig growth. Therefore, to address the concerns and give a better understanding of the mechanism of high copper diet in restraining pig growth in different systems, this paper reviews the research progress of long-term supplementation of high copper on the growth of pigs and provides some suggestions and further research directions.

How Can Xenosensors Act in Chemical Detoxification Metabolism?

There are some disparities between pharmacological and toxicological xenobiotic receptor (xenosensors) pathways. These variations include receptor models that indicate several toxic patterns. Such models have demanded some update from traditional medical receptor relations studied by pharmacologists. These may include the response time, the molecular level, and unclear directions of toxicological metabolism. Xenosensors activities were affected by many factors that include genetic elements, physiological status, xenobiotic complication, and species-specific variations. Thus, this review aims to highlight the most advanced features of xenosensors related to toxicant biotransformations and other patterns such as characteristics, recognition, and the relations between different xenosensors.

LPS-induces IL-6 and IL-8 gene expression in bovine endometrial cells "through DNA methylation".

Uterine infection in dairy cows causes great economic loss. In bovine endometrial cells, lipopolysaccharide (LPS)-stimulated increase in interleukin 6 (IL-6) and interleukin 8 (IL-8) mRNA is crucial for the inflammatory response; however, the regulatory mechanisms remain unclear. Here, we investigated the role of DNA methylation in IL-6 and IL-8 mRNA expression following LPS-induction in bovine endometrial cells. IL-6 and IL-8 mRNA expression was evaluated under DNA methylation inhibition using 5-Aza-2'-deoxycytodine (5Aza) following LPS stimulation. Expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), methyl CpG-binding protein 2 (MeCP2) and DNA methylation at IL-6 and IL-8 regions, were analyzed using quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR (BSP) following 24 h of LPS treatment. Inhibition of DNA methylation significantly enhanced LPS-induced IL-6 and IL-8 mRNA expression. LPS increased IL-6 and IL-8 mRNA expression, and decreased methylation levels of specific CpG sites at the IL-6 promoter (at -366 and -660) and the IL-8 promoter (at -120 and -48) after 24 h. Furthermore, LPS treatment for 24 h significantly increased DNMT1, DNMT3A, DNMT3B, and MeCP2 mRNA expression. Our results indicate that treating bovine endometrial cells with LPS induces the expression of IL-6 and IL-8 mRNA regulated by IL-6 and IL-8 promoter methylation.

Integrated analysis of miRNA and mRNA expression profiling in bovine endometrial cells in response to lipopolysaccharide-stimulation.

MicroRNAs (miRNA) play an important role in regulating gene expression, making them important resources for exploring molecular mechanisms. Molecular mechanisms involved in the inflammatory responses of bovine endometrial cells induced by lipopolysaccharide (LPS) have not been widely studied. In the present study, miRNA and mRNA expression profiling of bovine endometrial cells treated with 1 µg/mL LPS for 24 h were evaluated by RNA-Seq (RNA-sequencing). The results showed that LPS induced 20 (11 up- and 9 down-regulated) differentially-expressed miRNAs and 108 (90 up- and 18 down-regulated) differentially-expressed mRNAs of bovine endometrial cells. The results for 5 mRNAs and 4 miRNAs were evaluated by quantitative real-time PCR (qRT-PCR) to validate the reliability of the RNA-seq data. Integrating analysis of the miRNA and mRNA expression profiles revealed 116 miRNA-target gene pairs. GO and KEGG pathway analysis of differentially expressed miRNAs and target genes predicted the likely roles of differentially expressed miRNAs in inflammatory responses in bovine endometrial cells induced by LPS. The reliability of the integrating analysis of the miRNA and mRNA data were validated by measuring the expression of three miRNA-target gene pairs by qRT-PCR. Our results improve the understanding of the role of miRNA involvement in inflammatory response of bovine endometrial cells induced by LPS.

Effects of dietary supplementation of Acanthopanax senticosus on gastrointestinal tract development in calves.

Twenty-four newborn Holstein dairy male calves (with initial body weight of 38 ± 3.0 kg) were used in a randomized block design experiment to determine effects of dietary supplementation of Acanthopanax senticosus (AS) on gastrointestinal tract development. Calves were fed milk (10%/body weight) three times at 06.00, 12.00 and 18.00 hours daily with one to four treatments during the experimental periods (4 to 28 days): no supplementation of AS (control group, CG); 1.0 g/L•time of micro-powder AS (MP); 1.0 g/L•time of superfine powder AS (SP); or 1.0 g/L•time of coarse powder AS (CP). On days 7, 14, 21 and 28, 20 mL blood samples were collected at 06.00 hours before the morning feeding. At the end of the trial (28 days), all calves were euthanized, and tissue samples were taken and placed in 4% buffered formaldehyde for analyses. In the rumen of MP treatment, compared with the CG treatment, wall thickness and papillae diameter was both significantly lower (P<0.05), while crypt depth was significantly greater (P<0.05). In the duodenum, villi diameter of AS supplemented treatments was significantly lower than that of CG treatment (P<0.05). Results indicate that calves during sucking period supplemented with AS as MP style could promote gastrointestinal development.

High Dietary Copper Increases Catecholamine Concentrations in the Hypothalami and Midbrains of Growing Pigs.

The experiment was conducted to investigate the effect of high dietary copper on catecholamine concentration and dopamine-ß-hydroxylase (DßH) activity in hypothalami and midbrains of growing pigs. Forty-five crossbred weanling pigs with an average body weight of 7.5 kg were randomly assigned to three groups of 15 each to receive a control diet containing 10 mg/kg Cu (diet A) and diets containing 125 (diet B) or 250 (diet C) mg Cu/kg DM for 45 days. Compared to the control, Cu supplementation at both 125 and 250 mg Cu/kg DM increased average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency. High dietary copper increased midbrain and hypothalami dopamine (DA) and norepinephrine (NE) concentrations and midbrain dopamine-ß-hydroxylase activity. However, increasing dietary Cu had no effect on hypothalami dopamine-ß-hydroxylase activity.

High lever dietary copper promote ghrelin gene expression in the fundic gland of growing pigs.

This experiment was conducted to examine the effect of dietary copper supplementation on ghrelin mRNA expression level in the fundic gland of growing pigs. A total of 45 crossbred pigs were randomly assigned to three groups of 15 pigs, five replicates of three animals comprised each group. Pigs were allocated to diets that contained 5 mg/kg copper (as the control group), 125 mg/kg copper sulfate, or 125 mg/kg copper methionine. At the end of the experiment, five pigs were selected at random from each group, slaughtered, and collected the fundic gland for determination of ghrelin mRNA expression level. The results showed that average daily gain, average daily feed intake, absolute weight, serum growth hormone (GH) concentration, and ghrelin mRNA level were higher in pigs fed the diets with 125 mg/kg copper methionine and 125 mg/kg copper sulfate (P < 0.05), than in pigs fed a diet with 5 mg/kg copper. These data suggest that high dietary copper (125 mg/kg) appears to increase feed intake and promote weight gain by enhancing the secretion of GH and ghrelin mRNA level in growing pigs.

Effect of high dietary copper on somatostatin and growth hormone-releasing hormone levels in the hypothalami of growing pigs.

This experiment was conducted to examine the effect of dietary copper supplementation on somatostatin (SS) and growth hormone-releasing hormone (GHRH) mRNA expression levels in the hypothalami of growing pigs. A total of 45 crossbred pigs were randomly assigned to three groups of 15 pigs each; five replicates of three animals comprised each group. Pigs were allocated to diets that contained 5 mg/kg copper (control), 125 mg/kg copper sulfate, or 125 mg/kg copper methionine. At the end of the experiment, five pigs were selected at random from each group and slaughtered, and hypothalami were collected for determination of SS and GHRH mRNA expression levels. The results showed that the SS expression levels were lower and the GHRH levels were higher in pigs fed the diets with 125 mg/kg copper methionine (P<0.05) and 125 mg/kg copper sulfate (P<0.05), respectively, than in pigs fed the diet with 5 mg/kg copper. Furthermore, the relationship between SS mRNA and GHRH mRNA abundance had a significantly negative correlation (P<0.05). The data indicated that high dietary copper could enhance GHRH mRNA expression levels and suppress SS mRNA expression levels in the hypothalami of pigs. High lever dietary copper (125 mg/kg copper sulfate or 125 mg/kg copper methionine) increased pigs' growth performance and feed efficiency but had no significant effect on daily feed intake; 125 mg/kg copper sulfate or 125 mg/kg copper methionine at the same lever had no difference on growth promoting in pigs.