Molecular Characterization of Microtubule Affinity-Regulating Kinase4 from Sus scrofa and Promotion of Lipogenesis in Primary Porcine Placental Trophoblasts.

This study aimed to characterize the full-length cDNA of MARK4 in Sus scrofa, and evaluated its potential role in the regulation of lipid accumulation in pig placental trophoblasts and analyzed signaling pathways involved, thereby providing insights into mechanisms for placental lipotoxicity induced by excessive back-fat during pregnancy of sows. The cDNA obtained with 5' and 3' RACE amplification covered 3216 bp with an open reading frame of 2259 bp encoding 752 amino acids. Multiple alignments and phylogenetic analysis revealed MARK4 protein of Sus scrofa had a high homology (95%⁻99%) to that of other higher vertebrates. After transfection, enhanced MARK4 significantly promoted lipogenesis in pig trophoblasts, as evidenced by accelerated lipid accumulation and consistently increased mRNA expressions of lipogenic genes DGAT1, LPIN1, LPIN3, LPL, PPARδ and SREBP-1c. Meanwhile, PPARγ remarkably inhibited the stimulating effect of MARK4 on non-receptor-mediated lipid accumulation in trophoblasts. Further analyses revealed WNT signaling enhanced lipid accumulation and activation of MARK4 in pig trophoblast cells. Finally, we demonstrated that WNT/ß-catenin signal pathway is involved in MARK4 activated lipogenesis. These results suggest that MARK4 promotes lipid accumulation in porcine placental trophoblasts and can be considered as a potential regulator of lipotoxicity associated with maternal obesity in the pig placenta.

Effects of four short-chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage.

BACKGROUND: Short-chain fatty salts have been widely used as food and forage preservatives because of their antimicrobial properties. This study evaluated the effects of four chemical compounds with antimicrobial properties on nitrogen transformations and intrinsic protease activity of alfalfa silage. RESULTS: Potassium diformate (PD) and formic acid (FA) rapidly reduced silage pH. Silages treated with sodium diacetate (SD) and calcium propionate (CAP) had higher final peptide N concentrations than other silage. The free amino acid N contents in PD and FA treated silages were lower than other silages at all intervals of ensilage. The ammonia N concentrations in FA and PD silages were the lowest, followed by SD and CAP silages. As ensiling progressed, the aminopeptidase activity was completely lost by day 5 for FA and PD silages and inactive by day 7 for SD silage, while it remained active after day 7 for control and CAP silage. The carboxypeptidase activities in FA and PD silages were already reduced below 50% by day 1 of ensiling. CONCLUSION: Potassium diformate was as effective as formic acid in depressing the proteolysis, while sodium diacetate and calcium propionate were inferior to formic acid in protecting alfalfa proteins from being hydrolysed. © 2016 Society of Chemical Industry.

Effects of sodium diacetate on the fermentation profile, chemical composition and aerobic stability of alfalfa silage.

OBJECTIVE: The objective of this study was to evaluate the effect of sodium diacetate (SDA) on fermentation profile, chemical composition and aerobic stability of alfalfa (Medicago sativa L.) silage. METHODS: Fresh alfalfa was ensiled with various concentrations of SDA (0, 3, 5, 7, and 9 g/kg of fresh forage). After 60 days of the ensiling, the samples were collected to examine the fermentative quality, chemical composition and aerobic stability. RESULTS: The application of SDA significantly (p<0.05) decreased silage pH with the lowest value in silage with 7 g/kg of SDA. The proliferations of enterobacteria, yeasts, molds and clostridia were inhibited by SDA, resulted in lower ethanol, propionic and butyric acid concentrations and dry matter loss in SDA treated silages than control. The increasing SDA linearly decreased free amino acid N (p<0.001), ammonia N (p = 0.018) and non-protein N (p<0.001), while linearly increased water soluble carbohydrate (p<0.001) and peptide N (p<0.001). It is speculated that SDA accelerated the shift from homofermentative to heterofermentative lactic acid bacteria during the silage fermentation, indicated by lower lactic acid production in SDA-9 than SDA-7 silages after 60 days of ensiling. Alfalfa silages treated with SDA at 7 g/kg had highest Flieg's point and remained stable more than 9 d during aerobic exposure under humid and hot conditions in southern China. CONCLUSION: SDA may be used as an additive for alfalfa silages at a level of 7 g/kg.

Effects of calcium propionate on the fermentation quality and aerobic stability of alfalfa silage.

OBJECTIVE: To assess the potency of calcium propionate (CAP) used as silage additive, an experiment was carried out to evaluate the effect of CAP on the nitrogen transformation, fermentation quality and aerobic stability of alfalfa silages. METHODS: Alfalfa was ensiled with four levels of CAP (5, 10, 15, and 20 g/kg of fresh weight [FW]) in laboratory silos for 30 days. After opening, the silages were analyzed for the chemical and microbiological characteristics, and subjected to an aerobic stability test. RESULTS: The increasing proportion of CAP did not affect pH, lactic acid (LA) concentrations and yeast counts, while linearly decreased counts of enterobacteria (p = 0.029), molds (p<0.001) and clostridia (p<0.001), and concentrations of acetic acid (p<0.001), propionic acid (p<0.001), butyric acid (p<0.001), and ethanol (p = 0.007), and quadratically (p = 0.001) increased lactic acid bacteria counts. With increasing the proportion of CAP, the dry matter (DM) loss (p<0.001), free amino acid N (p<0.001), ammonia N (p = 0.004), and non-protein N (p<0.001) contents were linearly reduced, whereas DM (p = 0.048), water soluble carbohydrate (p<0.001) and peptide N (p<0.001) contents were linearly increased. The highest Flieg's point was found in CAP10 (75.9), represented the best fermentation quality. All silages treated with CAP improved aerobic stability as indicated by increased stable hours compared with control. CONCLUSION: The addition of CAP can suppress the undesirable microorganisms during ensiling and exposure to air, thereby improving the fermentation quality and aerobic stability as well as retarding the proteolysis of alfalfa silage. It is suggested that CAP used as an additive is recommended at a level of 10 g/kg FW.

Interactive effects of glutamine and gamma-aminobutyric acid on growth performance and skeletal muscle amino acid metabolism of 22-42-day-old broilers exposed to hot environment.

The present experiment was conducted to investigate the interactive effects between dietary glutamine (Gln, 0 and 5 g/kg) and gamma-aminobutyric acid (GABA, 0 and 100 mg/kg) on growth performance and amino acid (AA) metabolism of broilers under hot environment. A total of 360 22-day-old Arbor Acres male chickens were randomly assigned to five treatment groups under thermoneutral chamber (PC, 23 °C) and cyclic heat stress (HS, 30-34 °C cycling) conditions. Compared with the PC group, cyclic HS decreased (P < 0.05) daily weight gain (DWG), daily feed consumption (DFC), the concentrations of Gln, glutamate (Glu), and GABA, and the activities of glutaminase and glutamic acid decarboxylase (GAD) in breast muscle at 28, 35, and 42 days, while it increased (P < 0.05) the activities of glutamine synthetase (GS) and gamma-aminobutyric acid transaminase (GABA-T) at 28, 35, and 42 days. Dietary Gln and GABA improved (P < 0.05) DWG and DFC of broilers under cyclic HS during 28-42 days. In breast muscle, the Gln supplementation increased (P < 0.05) the concentrations of Gln (28, 35, and 42 days), Glu (28, 35, and 42 days), and GABA (42 days) and the activities of glutaminase (28, 35, and 42 days) and GAD (28, 35, and 42 days) but decreased (P < 0.05) GS activities at 28, 35, and 42 days and GABA-T activities at 28 days. The addition of GABA increased (P < 0.05) the concentrations of Gln and Glu and activities of glutaminase and GAD, while it decreased (P < 0.05) GABA-T activities at 28, 35, and 42 days. Significant interactions (P < 0.05) between Gln and GABA were found on breast skeletal muscle Gln concentrations, glutaminase activities, GS activities at 28 and 35 days, and DWG, GABA concentrations, and GABA-T activities at 28, 35, and 42 days in broilers under cyclic HS. In conclusion, the present results indicated that the interactions of exogenous Gln and GABA could offer a potential nutritional strategy to prevent HS-related depression in skeletal muscle Gln and GABA metabolism of broilers.

Selenium-Rich Black Soldier Fly Supplementation Enriches Serum Indexes and Egg Selenium Content in Laying Hens.

A certain amount of selenium (Se) is usually added to the diet of laying hens to improve the quality and nutritional value of eggs. The present study was carried out to investigate the effect of selenium-rich black soldier fly (BSF) supplementation in diets on laying production performance, egg quality, serum indexes, and egg selenium content of Hy-line variety brown laying hens. A total of 288 at 49-week-old healthy laying hens were divided into 3 treatment groups with 6 replicates per group and 16 hens per replicate using a single-factor completely randomized design. Treatments consisted of (1) control (basal diet without supplemental Se), (2) 0.30 mg/kg supplemental Se, (Se as sodium selenite, SS), and (3) 0.30 mg/kg supplemental Se (Se as selenium-rich black soldier fly, SE-BSF). Laying performance was not affected by dietary Se. There was no effect of selenium-rich BSF on egg quality (P > 0.05). The contents of malonaldehyde (MDA) were significantly reduced (P < 0.05). On the contrary, dietary Se supplementation increased the activity of superoxide dismutase (SOD, P < 0.05) and catalase (CAT, P < 0.05) and increased the concentration of reduced glutathione (P < 0.05). In addition, selenium-rich BSF supplementation significantly increased the Se content of eggs (P < 0.05). These results indicate that Se supplementation did not affect laying production performance and egg quality of laying hens, but the supplementation could improve antioxidant capacity and increased the Se content of eggs.

Berberine alleviates ETEC-induced intestinal inflammation and oxidative stress damage by optimizing intestinal microbial composition in a weaned piglet model.

Introduction: Enterotoxigenic Escherichia coli (ETEC) is the main diarrhea-causing pathogen in children and young animals and has become a global health concern. Berberine is a type of "medicine and food homology" and has a long history of use in China, particularly in treating gastrointestinal disorders and bacterial diarrhea. Methods: In this study, we explored the effects of berberine on growth performance, intestinal inflammation, oxidative damage, and intestinal microbiota in a weaned piglet model of ETEC infection. Twenty-four piglets were randomly divided into four groups-a control group (fed a basal diet [BD] and infused with saline), a BD+ETEC group (fed a basal diet and infused with ETEC), a LB+ETEC group (fed a basal diet with 0.05% berberine and infused with ETEC infection), and a HB+ETEC group (fed a basal diet with 0.1% berberine and infused with ETEC). Results: Berberine significantly improved the final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) (P<0.05) of piglets, and effectively decreased the incidence of diarrhea among the animals (P<0.05). Additionally, berberine significantly downregulated the expression levels of the genes encoding TNF-α, IL-1ß, IL-6, IL-8, TLR4, MyD88, NF-κB, IKKα, and IKKß in the small intestine of piglets (P<0.05). ETEC infection significantly upregulated the expression of genes coding for Nrf2, CAT, SOD1, GPX1, GST, NQO1, HO-1, GCLC, and GCLM in the small intestine of the animals (P<0.05). Berberine significantly upregulated 12 functional COG categories and 7 KEGG signaling pathways. A correlation analysis showed that berberine significantly increased the relative abundance of beneficial bacteria (Gemmiger, Pediococcus, Levilactobacillus, Clostridium, Lactiplantibacillus, Weissella, Enterococcus, Blautia, and Butyricicoccus) and decreased that of pathogenic bacteria (Prevotella, Streptococcus, Parabacteroides, Flavonifractor, Alloprevotella) known to be closely related to intestinal inflammation and oxidative stress in piglets. In conclusion, ETEC infection disrupted the intestinal microbiota in weaned piglets, upregulating the TLR4/MyD88/NF-κB and Nrf2 signaling pathways, and consequently leading to intestinal inflammation and oxidative stress-induced damage. Discussion: Our data indicated that berberine can optimize intestinal microbiota balance and modulate the TLR4/MyD88/NF-κB and Nrf2 signaling pathways, thus helping to alleviate intestinal inflammation and oxidative damage caused by ETEC infection in weaned piglets.

Identification and characterization of extrachromosomal circular DNA in Wei and Large White pigs by high-throughput sequencing.

Wei pig (WP) and Large White pig (LP) are fatty and lean breeds, respectively. Extrachromosomal circular DNA (eccDNA) plays an important role in regulating signaling pathway processes of cell. However, there are few reports regarding the eccDNA and ecDNA profiles in WP and LP. The present work aimed to investigate the eccDNA and ecDNA profiles between WP and LP. Three WPs and three LPs (100 ± 1.3 kg) were selected for analysis of eccDNA and ecDNA in the ear samples. Results showed that there were 39,686,953,656-58,411,217,258 and 53,824,168,657-58,311,810,737 clean data for WP and LP, respectively. Sequencing yielded 15,587-25,479 and 71,123-79,605 eccDNAs from the ear samples of WP and LP, respectively. There were 15,111 and 22,594 eccDNA-derived genes in the WP and LP, respectively, and 13,807 eccDNA-derived genes were common in the ear samples of both pigs. Sequencing yielded 13-19 and 27-43 ecDNAs in the ears of WP and LP, respectively. There were 1,005 and 1,777 ecDNA-derived genes in WP and LP, respectively, and 351 ecDNA-derived genes were common in the ear samples of both pigs. The most significant KEGG pathways of eccDNA-derived genes were axon guidance, focal adhesion, metabolic pathways, MAPK signaling pathway, Hedgehog signaling pathway, microRNAs in cancer, tight junction, phospholipase D signaling pathway, endocytosis, and sphingolipid signaling pathway. Furthermore, the most significant KEGG pathways of ecDNA-derived genes were olfactory transduction, B cell receptor signaling pathway, and chemical carcinogenesis. The eccDNA00044301 was lower abundance, while the ecDNA00000060 was higher abundance in WP compared with that in LP. Summary, we found that eccDNAs and ecDNAs are common in WP and LP and occur in sizes large enough to carry one or several partial or complete genes. These findings have expanded the knowledge repertoire of circular DNA in pig and will provide a reference for the use of pigs as a medical model and help discovery of new genetic markers to select high-quality breeds.

Genes and Signaling Pathways Involved in the Regulation of Selenium-Enriched Yeast on Liver Metabolism and Health of Broiler (Gallus gallus).

Selenium-enriched yeast (SeY) plays an important role in the liver health and metabolism of the broiler. However, the mechanism by which it regulates liver metabolism and the health of broilers is largely unknown. Therefore, this study was conducted to elucidate the key genes and signaling pathways involved in regulating SeY in liver metabolism and bird's health. Thus, the mRNA expression microarray, GSE25151, was downloaded from Gene Expression Omnibus (GEO) database. GSE25151 consists of liver samples from SeY-treated and the control broilers. Six hundred four differentially expressed genes (DEGs) were identified in livers between SeY-treated and control. Gene ontology (GO) enrichment analysis indicated that those DEGs are mainly involved in metabolism-related biological processes, such as biological regulation, molecular processes, responses to stimuli, cell communication and proliferation, and growth. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the DEGs mainly enriched in metabolism-related signaling pathways, including PI3K, Akt, Wnt, calcium, IGF1 receptor, and MAPK signaling pathways. Moreover, many genes, such as NMUR1, NMU, and GPRC6A, might contribute to the regulation of SeY to broiler liver metabolism and health. In conclusion, the current study enhances our understanding of the regulation of SeY in liver metabolism and health of the birds and will assist studies of the molecular mechanisms of SeY regulation in chicken liver.

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin.

Background: Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-ß superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole. Methods: Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-ß superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA. Results: Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin. Conclusion: High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Luzindole and 4P-PDOT block the effect of melatonin on bovine granulosa cell apoptosis and cell cycle depending on its concentration.

Granulosa cells play an essential physiological role in mediating the follicle development and survival or apoptosis of granulosa cells dictate the follicle development or atresia. The aim of this study was to investigate the role of high dose (10-5 M) and low dose (10-9 M) melatonin in bovine granulosa cells, and assess whether MT1 and MT2 inhibiter affect granulosa cells response to melatonin. We found that the high dose (10-5 M) and low dose (10-9 M) both could act as an essential role in modulating granulosa cells apoptosis, cell cycle and antioxidant. The beneficial effect could be related to that melatonin promoted the expression of Bcl2, Bcl-xl, SOD1 and GPX4, and inhibited Bax, caspase-3 and p53 expression. Moreover P21 expression was decreased in granulosa cells treated with the high dose (10-5 M) melatonin and increased in that treated with the low dose (10-9 M) melatonin. To further reveal the role of MT1 and MT2 in mediating the effect of melatonin on granulosa cells apoptosis, cell cycle and antioxidant, we found that the luzindole and 4P-PDOT did not affect the effect of high dose (10-5 M) melatonin on regulating Bcl2, Bax, caspase-3, SOD1, GPX4 and p53 expression, while blocked its effect on modulating Bcl-xl and P21expression. However, luzindole and 4P-PDOT disturbed the effect of low dose (10-9 M) melatonin on regulating Bcl2, Bax, caspase-3, Bcl-xl, SOD1, GPX4, and p53 expression. In conclusion, these results reveal that the effect of low dose (10-9 M) melatonin on granulosa cells apoptosis are mediated by MT1 and MT2, and the high dose (10-5 M) melatonin affect the granulosa cells apoptosis by other pathway, besides MT1 and MT2. Moreover MT1 and MT2 may work in concert to modulate bovine granulosa cells function by regulating cellular progression and apoptosis.

Impaired Mitochondrial Function Results from Oxidative Stress in the Full-Term Placenta of Sows with Excessive Back-Fat.

The aim of this study was to determine the effect of excessive back-fat (BF) of sows on placental oxidative stress, ATP generation, mitochondrial alterations in content and structure, and mitochondrial function in isolated trophoblasts. Placental tissue was collected by vaginal delivery from BFI (15-20 mm, n = 10) and BFII (21-27 mm, n = 10) sows formed according to BF at mating. Our results demonstrated that excessive back-fat contributed to augmented oxidative stress in term placenta, as evidenced by excessive production of ROS, elevated protein carbonylation, and reduced SOD, GSH-PX, and CAT activities (p < 0.05). Indicative of mitochondrial dysfunction, reduced mitochondrial respiration in cultured trophoblasts was linked to decreased ATP generation, lower mitochondrial Complex I activity and reduced expression of electron transport chain subunits in placenta of BFII sows (p < 0.05). Meanwhile, we observed negative alterations in mitochondrial biogenesis and structure in the placenta from BFII group (p < 0.05). Finally, our in vitro studies showed lipid-induced ROS production resulted in mitochondrial alterations in trophoblasts, and these effects were blocked by antioxidant treatment. Together, these data reveal that excessive back-fat aggravates mitochondrial injury induced by increased oxidative stress in pig term placenta, which may have detrimental consequences on placental function and therefore impaired fetal growth and development.

Glutamine improves heat stress-induced oxidative damage in the broiler thigh muscle by activating the nuclear factor erythroid 2-related 2/Kelch-like ECH-associated protein 1 signaling pathway.

The aim of the present study was to evaluate the effect of glutamine (Gln) on modulating heat stress-induced oxidative damage in the broiler thigh muscle through nuclear factor erythroid 2-related 2/Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway. Three-hundred 22-day-old Arbor Acres broilers were reallocated into 5 groups: a control group (24 °C) fed with basal diet and 4 heat stress (HS) groups (34 °C for 8 h/D) fed with basal diet containing 0, 0.5, 1.0, and 1.5% Gln. This experiment lasted 21 D. Heat stress decreased (P < 0.05) pH, redness, and Gln levels, and increased (P < 0.05) luminance, water loss rate, and cooking loss (CL) values of the thigh meat. Compared with the HS group, supplementation with 1.5% Gln increased (P < 0.05) pH, redness, and Gln levels, but decreased (P < 0.05) luminance and CL values in the thigh meat. There were significant decreases (P < 0.05) in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and Nrf2 levels, but significant increases (P < 0.05) in the malondialdehyde (MDA) and Keap1 levels of the thigh muscle after HS treatment. Compared with the HS group, supplementation with 1.0, and 1.5% Gln decreased (P < 0.05) MDA and Keap1 levels; supplementation with 1.5% Gln increased (P < 0.05) GSH, GSH-Px, T-AOC, CAT, SOD, and Nrf2 levels in the thigh muscle of heat-stressed broilers. Furthermore, HS decreased (P < 0.05) Nrf2, SOD, CAT, and GSH-Px mRNA expression levels, but increased (P < 0.05) Keap1 mRNA level in the thigh muscle of broiler. Dietary supplementation with 1.5% Gln increased (P < 0.05) Nrf2, GSH-Px, CAT, and SOD mRNA expression levels, but decreased (P < 0.05) Keap1 mRNA level in the thigh muscle of heat-stressed broilers. In conclusion, dietary Gln improved the resistance of heat-stressed broiler muscles to oxidative damage possibly through reversing the muscle Gln level and inducing the expression of the Nrf2-Keap1 pathway.

Efficient Expression of Xylanase by Codon Optimization and Its Effects on the Growth Performance and Carcass Characteristics of Broiler.

The aim of the present study was to improve the expression level of Trichoderma reesei xylanase (XynB) in Pichia pastoris through a codon optimization strategy and evaluate its effects on the growth performance and carcass characteristics of broiler. According to the codon bias of Pichia genome, the XynB gene from T. reesei was optimized and synthesized by whole gene assembly to improve its expression level in P. pastoris. Approximately 180 target mutations were successfully introduced into natural XynB. The maximum activity of xylanase (optiXynB) secreted by P. pastoris pPICZaA-optiXynB was 1299 U/mL after 96 h induction. Purified recombinant optiXynB had the molecular weight of 24 kDa. The optiXynB presented highest activity in pH 5.0 and 50 °C. The recombinase was highly specific towards birchwood xylan, beechwood xylan, and oat-spelt xylan. In the broiler experiment, a total of 200 Arbor Acre broilers (one day old) were randomly allocated into four groups fed with basal diets containing 0 (control group), 500, 1000, and 1500 IU/kg optiXynB. Dietary 1000 and 1500 IU/kg optiXynB significantly increased (p < 0.05) final weight and body weight gain; dietary 500, 1000, and 1500 IU/kg optiXynB significantly increased (p < 0.05) pre-evisceration weight, dressed percentage, and eviscerated weight compared with the control group. Inclusion of optiXynB in broiler diets linearly increased final weight, body weight gain, breast muscle weight and leg muscle weight, but linearly decreased feed conversion rate (p < 0.05). Furthermore, inclusion of optiXynB in broiler diets linearly and quadratically increased pre-evisceration weight, dressed percentage, and eviscerated weight (p < 0.05). The recombinant optiXynB from P. pastoris pPICZaA-optiXynB was beneficial in improving growth performance and carcass characteristics of broilers.

Influence of Dietary Copper Methionine Concentrations on Growth Performance, Digestibility of Nutrients, Serum Lipid Profiles, and Immune Defenses in Broilers.

A 42-day experiment was conducted to evaluate the influence of dietary copper (Cu) concentrations on growth performance, nutrient digestibility, and serum parameters in broilers aged from 1 to 42 days. Five hundred forty 1-day-old broilers were randomly assigned into 1 of the following 6 dietary treatments: (1) control (basal diet without supplemental Cu), (2) 15 mg/kg supplemental Cu (Cu15), (3) 30 mg/kg supplemental Cu (Cu30), (4) 60 mg/kg supplemental Cu (Cu60), (5) 120 mg/kg supplemental Cu (Cu120), and (6) 240 mg/kg supplemental Cu (Cu240), Cu as copper methionine. A 4-day metabolism trial was conducted during the last week of the experiment feeding. The results showed that dietary Cu supplementation increased the average daily gain and the average daily feed intake (P < 0.01). The feed gain ratio, however, was not affected by dietary Cu (P > 0.10). Additionally, dietary Cu supplementation increased the digestibility of fat and energy (P < 0.05). The concentration of serum cholesterol, triglycerides, and high-density lipoprotein cholesterol decreased with dietary Cu supplementation (P < 0.05). The activities of serum Cu-Zn superoxide dismutase (P < 0.05), glutathione peroxidase (P < 0.05), and ceruloplasmin (P = 0.09), on the contrary, were increased by Cu addition. For immune indexes, dietary Cu supplementation increased serum IgA and IgM (P < 0.05). In addition, the activities of serum ALT increased with increasing dietary Cu supplementation (P < 0.05). In conclusion, our data suggest that Cu supplementation can increase fat digestibility and promote growth. Additionally, dietary Cu supplementation can reduce serum cholesterol and enhance antioxidant capacity in broilers.

Cloning, expression and characterization of a cold-adapted endo-1, 4-ß-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis.

BACKGROUND: Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study, Citrobacter farmeri A1 which was isolated from a wood-inhabiting termite Reticulitermes labralis could secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml). The objective of the present study was to clone and express the C. farmeri EglC gene in Escherichia coli to improve production level and determine the enzymatic properties of the recombinant enzyme. METHODS: The EglC gene was cloned from C. farmeri A1 by thermal asymmetric interlaced PCR. EglC was transformed into vector pET22b and functionally expressed in E. coli. The recombination protein EglC22b was purified for properties detection. RESULTS: SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of the E. coli pET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5-8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30-40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co2+ and Fe3+, but inhibited by Cd2+, Zn2+, Li+, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA. CONCLUSION: These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications.

Effects of ethanol, molasses and Lactobacillus plantarum on the fermentation quality, in vitro digestibility and aerobic stability of total mixed ration silages in the Tibetan plateau of China.

In Tibet, it is common practice to make and relocate total mixed ration (TMR) silages before feeding due to the uneven distribution of forages temporally and spatially. This study was conducted to investigate the effects of Lactobacillus plantarum (L), molasses (M) or ethanol (E) on the fermentation quality and aerobic stability of local adaptive TMR silage. After 45 days of ensiling, pH and ammonia nitrogen in inoculated TMR silages were significantly lower than control. During the first 6 days of the aerobic exposure test, a small fluctuation in lactic acid concentration for all TMR silages was observed, and then silages with ethanol continued this trend, while lactic acid in silage without ethanol sharply decreased until the end of the aerobic exposure period. Meanwhile, pH gradually increased along the aerobic exposure; silages treated with ethanol showed lower pH after 9 days of aerobic exposure. The population of yeast gradually increased during 6 days of aerobic exposure, after that an accelerated rise was observed in TMR silages without ethanol. The combinational beneficial effect of L. plantarum and ethanol was found in combined addition of ethanol and Lactobacillus plantarum silages (EL), indicated by intermediate fermentation quality and higher aerobic stability.

Efficient expression and characterization of a cold-active endo-1, 4-β -glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.

Adding distiller's grains and molasses on fermentation quality of rice straw silages / Adição de melaço na qualidade de fermentação de silagem de palha de arroz

ABSTRACT: Ensilage is a simple and low-cost strategy to enable long term preservation and environmentally friendly utilization of agricultural by-products, such as straws and distiller's grains (DG) for ruminants. Effect of mixing different proportions of DG and rice straw (i.e. 0, 10, 20 or 30% of DG) with or without 5% molasses addition on fermentation and chemical variables of silages was evaluated. The study was conducted as a randomized blocks design in a 4 × 2 factorial arrangement, with three replications, using laboratory silos of 1L capacity (n=24). Despite a significant interaction (P<0.01) between DG and molasses addition was observed for most variables, in general the increased addition of DG linearly decreased the pH value, acetic acid (AA), butyric acid (BA) and ammonia N concentration (P<0.01), and increased the lactic acid (LA) concentration (P<0.01). Exception was the propionic acid concentration which linearly decreased without molasses addition and linearly increased with molasses addition at increased proportion of DG (P<0.01). In both silages with or without molasses the addition of DG increased the dry matter, water soluble carbohydrates and crude protein (P<0.01), and decreased the NDF content (P<0.01). Based on the perspective of maximum utilization of rice straw, the mixture of 10% of DG associated to 5% molasses at ensilage process is recommended.

RESUMO: Ensilagem é uma estratégia simples e de baixo custo que habilita a preservação de sub-produtos agrícolas por longo tempo e com mínimo impacto ambiental, tal como a preservação de palha de arroz e resíduos da destilação de grãos (DG) para uso na alimentação de ruminantes. O objetivo deste estudo foi avaliar o efeito de incluir diferentes proporções de DG e palha de arroz (i.e. 0, 10, 20 or 30% of DG) com ou sem inclusão de 5% de melaço sobre variáveis da fermentação e composição química do material ensilado. O estudo foi conduzido em blocos casualizados em um esquema fatorial 4 × 2, com três repetições, utilizando mini-silos de 1L de capacidade (n=24). Embora a interação entre DG e melaço foi significativa (P<0,01) para a maior parte das variáveis, em geral a adição de DG diminuiu linearmente o pH e as concentrações de ácido acético, ácido butírico e amônia (P<0,01), e aumentou linearmente a concentração de ácido láctico (P<0,01). Exceção foi a concentração de ácido propiônico que diminuiu linearmente sem a adição de melaço enquanto aumentou linearmente com a adição de melaço, à incrementados níveis de inclusão de DG (P<0,01). Em ambos casos, com ou sem adição de melaço, a adição de DG aumentou linearmente o teor de matéria seca, de carboidratos solúveis em água e de proteína bruta, e diminuiu o teor de fibra em detergente neutro do material ensilado the NDF content (P<0,01). Baseado na perspectiva de máxima utilização de palha da arroz, recomenda-se a mistura de 10% de DG associado com 5% de melaço no processo de ensilagem.