Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1).

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10-3 (S-1), and kcat/Km 62.77 (M-1 S-1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

Prediction of B cell and T-helper cell epitopes candidates of bovine leukaemia virus (BLV) by in silico approach.

The bovine leukaemia virus (BLV) is a retrovirus responsible for enzootic bovine leukaemia (EBL) disease, the most common cattle disease leading to high annual economic losses to the cattle breeding industry. Virus monitoring among the sheep and cattle herds is usually done by vaccination. Inactivated virus vaccines can partially protect the livestock from viral challenge. However, vaccinated animals are likely to be infected. So, there is an essential need for producing vaccine by other methods. Gp60 SU, encoded by Env gene, is the surface glycoprotein of BLV detected to be the major target for the host immunity against the virus. Different stages were performed to predict the potential B and T-helper cell epitopes. The general framework of the method includes retrieving the amino acid sequence of gp60 SU, conducting the sequence alignment, getting the entropy plot, retrieving the previously found epitopes, predicting the hydropathy parameters, modelling the tertiary structure of the glycoprotein, minimizing the structure energy, validating the model by Ramachandran plot, predicting the linear and discontinuous epitopes by various servers and eventually choosing the consensus immunogenic regions. Ramachandran plot scrutiny has demonstrated that the modelled prediction is accurate and suitable. By surveying overlaps of various results, 4 and 2 immunogenic regions were selected as linear and conformational epitopes respectively. Amino acids 35-53, 67-97, 288-302 and 410-421 and those of numbers 37-58 and 72-100 were the regions selected as linear and conformational epitopes respectively. The tertiary structure of the final epitope was modelled as well. A comparison of the predicted epitopes structure with that of gp60 SU envelope, illustrated that the tertiary structure of these epitopes does not change after being separated from the primary complete one. The present achievements will lead to a better interpretation of the antigen-antibody interactions against gp60 in the designing process of safe and efficient vaccines.

The effect of different levels of Fenugreek (Trigonella foenum-graecum L.) powder and extract on performance, egg quality, blood parameters and immune responses of laying hens in second production cycle.

The present study was carried out to determine the effects of fenugreek powder (FP) and extract (FE) on performance, egg quality, blood parameters and immune responses of laying hens. One-hundred and fifty Leghorn laying hens were used in a completely randomized design with five treatments and five replicates for eight weeks. Treatments were various levels of FP and FE including zero (control; T1), 1.00% FP (T2), 2.00% FP (T3), 0.10% FE (T4) and 0.20% FE (T5). The results of this experiment showed that feed intake was increased linearly by the inclusion of FP compared to the control group. Supplementation of laying hens diet with 2.00% FP adversely affected feed conversion ratio (FCR). The FCR was decreased by 0.10% inclusion of FE compared to 0.20%. Egg yolk color was the highest when 1.00% FP added to laying hens diets compared to the other treatments. Serum metabolites and immune responses of laying hens were not affected significantly by fenugreek supplementation. From the results of the present study, it can be concluded that using 1.00% FP can improve feed intake by supporting FCR. Inclusion of 1.00% FP in laying hens diet enhanced egg yolk color of laying hens in the second production cycle.

Reconstruction and Analysis of Cattle Metabolic Networks in Normal and Acidosis Rumen Tissue.

The objective of this study was to develop a system-level understanding of acidosis biology. Therefore, the genes expression differences between the normal and acidosis rumen epithelial tissues were first examined using the RNA-seq data in order to understand the molecular mechanisms involved in the disease and then their corresponding metabolic networks constructed. A number of 1074 genes, 978 isoforms, 1049 transcription start sites (TSS), 998 coding DNA sequence (CDS) and 2 promoters were identified being differentially expressed in the rumen tissue between the normal and acidosis samples (p < 0.05). The functional analysis of 627 up-regulated genes revealed their involvement in ion transmembrane transport, filament organization, regulation of cell adhesion, regulation of the actin cytoskeleton, ATP binding, glucose transmembrane transporter activity, carbohydrate binding, growth factor binding and cAMP metabolic process. Additionally, 111 differentially expressed enzymes were identified between the rumen epithelial tissue of the normal and acidosis steers with 46 up-regulated and 65 down-regulated ones in the acidosis group. The pathways and reactions analyses associated with the up-regulated enzymes indicate that most of these enzymes are involved in the fatty acid metabolism, biosynthesis of amino acids, pyruvate and carbon metabolism while most of the down-regulated ones are involved in purine and pyrimidine, vitamin B6 and antibiotics metabolisms. The degree distribution of both metabolic networks follows a power-law one, hence displaying a scale-free property. The top 15 hub metabolites were determined in the acidosis metabolic network with most of them involved in the fatty acid oxidation, VFA biosynthesis, amino acid biogenesis and glutathione metabolism which plays an important role in the stress condition. The limitations of this study were low number of animals and using only epithelial tissue (ventral sac) for RNA-seq.

Genome-wide identification and characterization of novel long non-coding RNA in Ruminal tissue affected with sub-acute Ruminal acidosis from Holstein cattle.

Sub-acute ruminal acidosis is a type of metabolic disorder in which affected cattle show a considerable depression of rumen pH. This leads to a dramatic decline in productivity and consequent loss of income for many dairy farms. The objective of the present study is to identify and characterize novel long non-coding RNAs (lncRNAs) in Holstein cattle affected by sub-acute ruminal acidosis. Two replicates from six animals were sequenced that bioinformatically analyzed. Results showed 6679 novel lncRNAs among which 12 intergenic lncRNAs showed differential expression (p value ≤0.05). GO and KEGG analysis revealed that calcium signaling and G protein couple-receptor pathways may be involved in regulating metabolic processes during sub-acute ruminal acidosis. Furthermore, other biological processes including transmembrane transport, adult behavior, neuroactive ligand-receptor interaction, GABAergic synapse, cholinergic synapse were significantly enriched. The present data suggest that these differentially expressed lncRNAs may play regulatory roles in modulating biological processes associated with sub-acute ruminal acidosis in cattle rumen.

Investigation of rumen fermentation parameters and some blood metabolites of dromedary camels fed with C3 and C4 forages.

The aim of this experiment was to investigate rumen fermentation and some blood parameters of dromedary camels fed with C3 and C4 forage. Four fistulated dromedary adult camels were fed with diets as a changeover design, 30 days for each period. The diets included alfalfa hay + wheat straw (C3 forage) and atriplex+ suaeda + seidlitzia (C4 forage). At the end of the experiment, rumen and blood parameters, gas production of wheat straw and atriplex as a 2 × 2 factorial experiment were determined. The highest blood glucose and urea nitrogen levels were found for camels fed with C3 forage, 2 hr after feeding (p < 0.05). The maximum NH3-N concentration in the rumen was for diets C3 and C4, 2 and 4 hr after feeding (p < 0.05). The lowest rumen pH was observed for C3 diet at 2 and 4 hr and for C4 diet at 4 and 8 hr after feeding. The activity of rumen carboxymethylcellulase (CMCase) and microcrystalline cellulase (MCCase) enzymes was the highest for C3 and C4 diets, 8 hr after feeding, however, during feeding the enzyme activity in C4 was higher than that of 2 hr (p < 0.05). The rumen volatile fatty acid (VFA) concentrations were significantly higher in camels fed C3 forage in comparison with C4 (p < 0.05). The results showed that the gas production potential was significantly higher in treatments containing atriplex, however, the gas production rate was higher in treatment containing wheat straw (p < 0.05). The results suggested that for camels maintained in closed systems, the replacement of C3 forages instead of C4 could be possible and useful.

RNA-Seq Bayesian Network Exploration of Immune System in Bovine.

BACKGROUND: The stress is one of main factors effects on production system. Several factors (both genetic and environmental elements) regulate immune response to stress. OBJECTIVES: In order to determine the major immune system regulatory genes underlying stress responses, a learning Bayesian network approach for those regulatory genes was applied to RNA-Seq data from a bovine leukocyte model system. MATERIAL AND METHODS: The transcriptome dataset GSE37447 was used from GEO and a Bayesian network on differentially expressed genes was learned to investigate the gene regulatory network. RESULTS: Applying the method produced a strongly interconnected network with four genes (TERF2IP, PDCD10, DDX10 and CENPE) acting as nodes, suggesting these genes may be important in the transcriptome regulation program of stress response. Of these genes TERF2IP has been shown previously to regulate gene expression, act as a regulator of the nuclear factor-kappa B (NF-κB) signalling, and to activate expression of NF-κB target genes; PDCD10 encodes a conserved protein associated with cell apoptosis; DDX10 encodes a DEAD box protein and is believed to be associated with cellular growth and division; and CENPE involves unstable spindle microtubule capture at kinetochores. Together these genes are involved in DNA damage of apoptosis, RNA splicing, DNA repairing, and regulating cell division in the bovine genome. The topology of the learned Bayesian gene network indicated that the genes had a minimal interrelationship with each other. This type of structure, using the publically available computational tool, was also observed on human orthologous genes of the differentially expressed genes. CONCLUSIONS: Overall, the results might be used in transcriptomic-assisted selection and design of new drug targets to treat stress-related problems in bovines.

Effect of radiation on chemical composition and protein quality of cottonseed meal.

This study was conducted to compare the effects of electron beam (EB) and gamma ray (GR) irradiation treatments at doses of 10, 20 and 30 kGy on chemical composition, protein quality and protein digestibility of cottonseed meal (CSM). GR irradiation in all doses significantly decreased the crude fiber of samples compared to raw CSM. Free and total gossypol content of CSM was decreased significantly by utilizing both types of irradiation in a dose-dependent manner. Also, EB irradiation caused decrease in free and total gossypol content more than that of GR irradiation. GR irradiation at doses of 20 and 30 kGy, and EB irradiation at doses of 10, 20 and 30 kGy can significantly decrease protein solubility of CSM compared to that of a raw sample. GR irradiation at a dose of 30 kGy significantly increased apparent digestibility of protein compared to raw and EB irradiation of CSM at a dose of 10 kGy in Leghorn cockerels. Maximum increase in protein digestibility of CSM was observed in GR irradiation at a dose of 30 kGy. In conclusion, the present study showed that EB and GR irradiation reduced gossypol and crude fiber and increased protein digestibility of CSM but had no effect on protein quality of CSM.

Inbreeding depression on growth traits of Iranian Lori sheep / Depresión endogámica en las características de crecimiento de la oveja Lori Iranní / Depressão endogâmica de características de crescimento em as ovelhas Lori Iraniana

Summary Background: Lori sheep is an Iranian heavy breed known for its superiority in terms of disease resistance, adaptability to the mountainous west of the country and meat production potential. Objective: to estimate and compare the inbreeding coefficient in Lori sheep, and its impact on growth traits. Methods: data and pedigree information were collected in Lorestan Agricultural and Natural Resources Research Center, west of Iran, during 2001-2010. Traits included were birth weight (BW), weaning weight (WW), 6-month weight (6MW) and 9-month weight (9MW). The inbreeding coefficient estimation was carried out through the CFC software, and quantification of individual inbreeding regression on the characteristics was conducted using The Wombat software. All animals were divided into four categories according to the inbreeding coefficients obtained from their pedigree: the first category included non-inbred animals (F = 0%); and the second, third, and fourth categories included inbred animals as 0<F ≤ 0.10, 0.10<F ≤ 0.20, and F>0.20, respectively. Results: inbreeding coefficients were 0.69% and 2.24% in the entire population and inbred population, respectively. Inbreeding regression for BW, WW, 6 MW and 9 MW were estimated as +4.5, -10.3, -76.3, and -77.4 g, respectively. The inbreeding trend was positive and significant for the whole population (0.215; p<0.001), but not significant for the inbred population. Conclusion: these results confirm a low level of inbreeding and suggest that direct controlled mating could be an appropriate method to avoid inbreeding depression.

Resumen Antecedentes: la oveja Lori es una de las razas pesadas Iraníes más conocidas por su superioridad en términos de resistencia a enfermedades, adaptabilidad a la zona montañosa del oeste del país y potencial para producción de carne. Objetivo: estimar y comparar el coeficiente de endogamia en la oveja Lori y su impacto sobre las características de crecimiento. Métodos: los datos y la información de pedigrí se recogieron en Lorestan Agricultural and Natural Resources Research Center, al oeste de Irán, desde los años 2001 al 2010. Los rasgos incluidos fueron peso al nacer (BW), peso al destete (WW), peso a los 6 meses (6 MW) y peso a los 9 meses (9 MW). La estimación del coeficiente de endogamia se llevó a cabo mediante el software CFC, y la cuantificación de la regresión de endogamia individual sobre las características se hizo con el software Wombat. Los animales fueron clasificados en cuatro categorías de acuerdo a los coeficientes de endogamia obtenidos de sus pedigrís; la primera categoría incluyó animales no endogámicos (F = 0%); la segunda, tercera y cuarta categorías incluyeron animales endogámicos como 0<F ≤ 0,10, 0,10<F ≤ 0,20 y F>0,20, respectivamente. Resultados: los coeficientes de endogamia fueron de 0,69 y 2,24% en toda la población y en la población endogámica, respectivamente. La regresión de endogamia para BW, WW, 6 MW, y 9 MW se estimó como +4,5, -10,3, -76,3 y -77,4 g, respectivamente. La tendencia endogámica fue positiva y significativa para toda la población (0,215; p<0,001), pero no significativa para la población endogámica. Conclusión: estos resultados confirman un bajo nivel de endogamia y sugieren que el apareamiento directo controlado podría ser un método apropiado para evitar la depresión endogámica.

Resumo Antecedentes: as ovelhas Lori são uma das raças pesadas iranianas conhecidas por sua superioridade comparativa sobre outras raças em termos de resistência a doenças, adaptabilidade ao montanhoso oeste do país e potencial para alta produção de carne. Objetivo: estimar e comparar o coeficiente de endogamia em ovelhas Lori e seu impacto sobre características de crescimento. Métodos: foram coletados dados e informações do pedigree no Lorestan Agricultural and Natural Resources Research Center, na província de Lorestan, oeste do Irã, entre 2001 e 2010. As características incluídas foram o peso ao nascimento (BW), o peso ao desmame (WW), o peso aos 6 meses (6MW), e o peso de 9 meses (9MW). A estimativa do coeficiente de endogamia foi realizada através de software CFC, e a quantificação da regressão de endogamia individual sobre as características foi levada a cabo através do software Wombat. Todos os animais foram classificados em quatro categorias de acordo com os coeficientes de endogamia obtidos por seu pedigree: a primeira categoria incluiu os animais não-endogâmicos (F = 0%); e a segunda, terceira e quarta categorias incluíram os animais endogâmicos como 0<F ≤ 0,10, 0.10<F ≤ 0,20 e F>0,20, respectivamente. Resultados: os coeficientes de endogamia foram de 0,69% e 2,24%, em toda a população e população consanguínea, respectivamente. A regressão da endogamia para BW, WW, 6 MW e 9 MW foi estimada em +4,5, -10,3, -76,3 e -77,4 g, respectivamente. A tendência da endogamia foi positiva e significativa em toda a população (0,215; p<0,001), mas não significativa na população consanguínea. Conclusão: os resultados confirmaram o baixo nível de endogamia e sugerem que a aplicação de um sistema de monta controlada direta poderia ser um método apropriado para evitar a depressão por endogamia.

Phenotypic and genetic parameter estimates for reproductive traits in Zandi sheep.

This study reports on the phenotypic and genetic (co)variance components for reproductive traits in Zandi sheep, using between 1,859 and 2,588 records obtained from 577 ewes. The data were collected from the Khojir Breeding Station of Zandi sheep in Tehran, Iran from 1994 to 2008. The basic traits were litter size at birth (LSB), litter size at weaning (LSW), litter mean weight per lamb born (LMWLB), and litter mean weight per lamb weaned (LMWLW), and the composite traits were total litter weight at birth (TLWB) and total litter weight at weaning (TLWW). Genetic analyses were carried out using the restricted maximum likelihood method that was explored by fitting the additive direct genetic effects and permanent environmental effects of the ewes as random effects and the ewe age at lambing and lambing year as fixed effects for all of the investigated traits. Akaike's information criterion was used to choose the most appropriate model. LSB, LSW, LMWLB, LMWLW, TLWB, and TLWW direct heritability estimates were 0.07, 0.05, 0.12, 0.10, 0.08, and 0.14, respectively. The estimated fractions of variance due to the permanent environmental effects of the ewe ranged from 0.03 for LMWLB to 0.08 for LMWLW and TLWW. Corresponding repeatability estimates ranged from 0.10 for LSW to 0.22 for TLWW. Direct genetic correlations varied from -0.61 for LSB-LMWLB to 0.88 for LSB-LSW and LSB-TLWB. Results indicate that genetic change depends not only on the heritability of traits, but also on the observed phenotypic variation; therefore, improvement of non-genetic factors should be included in the breeding programs.