Reference genes for livestock gene expression profiling – Literature review81-89Views:143
Quantitative real-time polymerase chain reaction (qPCR) is an essential tool for understanding animal cell’s response to developmental progression or to different experimental conditions at gene expression level. However the reliability of this method heavily lies on proper normalization (measuring a target and a reference gene’s expression from the same sample to correct for technical related variations).
Our literature review aimed to summarize the articles addressing the most important livestock species in regards of reference gene stability used as normalizers for quantitative real-time polymerase chain reaction experiments. Stably expressing reference genes were categorized into 14 distinct groups according to gene function. The number of reference genes tested and the publication numbers according to years and the ranking algorithms were also noted.
Counting showed that genes encoding ribosomal protein components are ranked as most stable in majority of cases and therefore should be taking into account for qPCR stable normalizer gene finding experiments.
Detection of DNA mutations by PCR-TTGE method21-25Views:161
In our study PCR-temporal temperature gelelectrophoresis (TTGE) and MeltINGENY bioinformatic program were used to analyse the mutations in the genes of melanocortin-1 receptor (MC1R) and pituitary adenylate-cyclase activating polypeptide (PACAP) in cattle. Amplification of target DNA by PCR was performed with GC-clamp primers and non-GC-clamp primers in simplex PCR reactions. The fragments were separated by denaturing polyacrylamide gelelectrophoresis (denaturing agents: high temperature, urea) after PCR reactions.MC1R homozygous individuals were used for the reaction.
We concluded that MeltINGENY program makes the decision and detection system easier, and more simple as the melting profile of target sequence is determined by the software. In case of MC1R gene, PCR-TTGE method is appropriate for SNP detection, however PACAP gene polymorphism can not be identified by the method, because PACAP mutations are not included in melting domains, therefore PCR-TTGE cannot detect them.
Role of hypothalamic neuropeptides in feed intake regulation of livestock species (literary review)63-68Views:174
Energy balance is the net result of the energy intake (nutrition) and expenditure (basic metabolic rate). The purpose of the daily feed intake is to provide energy and nutrients for maintenance, production and fill and maintain energy storages in form of glycogen and fat. Animals can adjust their feed intake to ensure their energy demand. Food intake regulation in animals and human is a very complex process, in which the digestive system, the central nervous system, the joining hormonal and non-hormonal factors, and the integrating hypothalamus take part. This review primarily focuses on the action mechanism of some important appetite regulating neuropeptides, and their impacts on the performance traits of the economically significant animal species.
Association analysis of TNNI1/XbaI polimorphism on carcass quality in hybrid pigs59-62Views:147
The contractile protein, which is encoded by troponin I 1 (TNNI1) gene, is located on the thin filaments of slow fibres in striated muscle. TNNI1 protein is a part of the troponin complex which plays an important role in regulation of muscle contraction by preventing actin-myosin interaction in absence of calcium. According to biological role, this gene can be potential marker for meat production related traits. The aim of this study is to define whether the previously reported gene polymorphism (EU743939:g.5174T>C) is connected with the slaughter traits measured in a standard slaughterhouse of the examined four-line European hybrid. The study included data from 404 gilts and barrows from 2 different samples. The polymorphism was detected using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method with XbaI restriction enzyme. In this study the allele frequencies were found as follows: C: 0.84 and 0.808; T: 0.16 and 0.192. Based on result of the present study no significant impact of polymorphisms on production parameters was found.
Associations analysis of production traits with leptin gene T3469C polymorphisms in pig39-43Views:136
The aim of this study was to define the connection between the leptin (LEP) gene T3469C polymorphism and its potential association with production traits in improved hybrid pigs. The study included data from 397 gilts and barrows from 2 different sample. The polymorphism was identified by using the polymerase chain reaction-restriction fragment-length polymorphism (PCR–RFLP) method with HinfI restriction enzyme. Two alleles of LEP gene were identified: T (0.93) an C (0.07). The analysis of values of production traits, depending on LEP genotype did not reveal significant (P≤0.05) differences. In the examination the loin diameter (between the 2nd and 3rd ribs), the live weight at slaughter and the averege daily gain during fattening were higher at pigs with C allele than pigs with TT genotipe. Accordingt to our data the effect of C allele was favourable in this population, because these animals had bigger bodyweight without valuable change of lean meat percent.
Search for polymorphism in exon 5 of cattle pituitary adenylate cyclase activating polypeptide gene17-20Views:152
Pituitary adenylate cyclase-activating polypeptide is a neuropeptide expressed primarily in the hypothalamus and in other tissues, which divergent and extensive physiological functions are proved. Large number of SNPs in PACAP are published involved SNP that is associated with phenotypic trait of cattle as well. Hungarian Grey, Hungarian Simmental, Holstein, Charolais and Angus cattle breeds were involved in this study to search for polymorphism in exon 5 1–391 bp and G/A transition. Our results of PCR RFLP and PCR SSCP did not prove the occurrence neither G/A transition, nor any other SNP in cattle breeds involved.
Influence of H-FABP gene polymorphisms on slaughter value of hybrid pigs55-60Views:206
The H-FABP gene was defined as a potential candidate gene influencing the fat deposition traits, primarily the intramuscular fat content. The aim of this study is to define whether the previously reported gene mutations are connected with the slaughter traits measured in a standard slaughterhouse. The study included data from 405 gilts and barrows from 2 different samples. The two chosen mutation (HFABP1: c. 103 T>C, HFABP2: c. 1970 T>C) were detected in one reaction with PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism) method with HinfI restrictoin enzyme. The allel frequencies are as follows: 103T(H)=0.75; 103C(h)=0.25, 1970T=0.32; 1970C=0.68. A HFABP1 mutation has significant effect on backfat thickness and lean meat % at stable 1 (sample 1), but there were no effect at stable 2 (sample 2). The analysis of values of production traits, depending on HFABP2 genotype did not reveal significant differences. Based on this study we can’t get a clear conclusion on the impact of polymorphisms on production parameters. In the examined flock the allele frequency of mutation in 5 'UTR is identical to the literature data, i. e. the more favorable variant regarding the intramuscular fat content is predominant in the population.
Fractionation of chicken egg proteome by isoelectric point in liquid phase39-42Views:157
The application of proteomics is relevant to physiology, reproduction, immunology, muscle and lactational biology in animal science, altough its use is still limited. One of the greatest challenges of proteome analysis is the reproducible fractionation of the complex protein mixtures. The fractionation methods can increase the probability of biomarker protein discovery. The fractionation by liquid-phase isoelectric focusing is one of the prefractionation methods. As a result, protein fractions can be easily collected, pooled and refractionated. There is a lack in the knowledge of gel-based proteomic methods of egg as only a limited number of protocols can be found in the literature, thus sample purification and fractionation require a time consuming optimisation procedure. The aim of this study was to fractionate egg yolk and white proteins by isoelectric point in liquid phase.
Identification of cow’s and buffalo’s milk and dairy product using a DNA-based method279-282Views:137
Aim of our study was the optimization of a DNA method, that is appropriate for reliable, low cost identification of animal species in milk and dairy product (cheese) and to determine the ratio of species. Mitochondrial DNA was used in our work to analyse buffalo/cow milk mixtures contained different ratio of bovine milk such as 0.1%, 0.5%, 1%, 1.5%, 2%, 5%, 10%, 15% (v/v%). Buffalo cheese were produced using buffalo and cows milk (0%, 2%, 5%, 10%, 15% – v/v% cows milk in buffalo milk). In case of milk mixtures, using species specific primers, the PCR assay showed a 0.5 v/v% detection limit. Cattle, in the buffalo/cows milk 99.9/0.1 v/v% mixture, was not detectable. The identification of buffalo and cows DNA in cheese was successful. The intensity of eletroforetic PCR fragment indicated the increase of cow milk ratio in milk and cheese samples as well.
The Effect of Grazing Intensities on Magnesium Contents8-13Views:156
Research was carried out on two areas of grassland in Hortobágy National Park, Hungary. Two herds of Hungarian Grey Cattle were kept in free range grazing and the effects of grazing pressure on the magnesium content of soil and ryegrass (Lolium perenne L) were determined.
Changes of plant available and total soil magnesium content under different grazing intensities did not show any evident tendency on the investigated grasslands. Different amounts of cattle faeces, urine and trampling had no effect on the magnesium concentration of ryegrass. We conclude that the magnesium content of ryegrass on both grassland sites as moderate grazing and overgrazing matches the requirements of cattle. Symptoms of magnesium deficiency of cattle will likely not appear.
Routine microscopy examination of faecal samples as a tool for detection of common gastrointestinal parasites: a preliminary report from two Hungarian farms63-66Views:72
Gastrointestinal parasitism in ruminant animals is a cause of major economic loss incurred by the livestock industry. Regardless of the frequency of the adopted therapeutic and prophylactic deworming strategies, the parasitic burden in a farm should be assessed regularly. One of the most widely used techniques to do so is the microscopic faecal egg examination and faecal egg counting method. Despite the technique being almost a century old from its first adoption, the principle behind the newer techniques of faecal egg examination is the same. This technique is still being used in routine farm screening and monitoring gastrointestinal parasitic load and faecal egg count reduction testing to assess the anthelmintic efficacy of the drugs used. Thus, the tool remains a choice for preliminary screening for important parasites and the subsequent deworming strategy. Our study here was part of a larger survey on the treatment efficiency as well as a broad epidemiological study of the trichostrongyle parasites in Hungary. We present a preliminary report on the detection of common gastrointestinal parasites from two farms in Hungary, including a species-specific confirmatory microscopy for Haemonchus contortus eggs.
Analysis of longevity in Holstein Friesian cattle using proteomic approaches21-25Views:123
The aim of the present study was to determine marker proteins those are associated with functional longevity of dairy cattle. Holstein-Friesian cows were grouped based on their performance as follows: group 1) individuals with good longevity traits; group 2) short production life because of poor reproduction traits; group 3) short production life with low milk yield. Twelve individuals were sampled in each group, blood and milk samples were collected from cows. Blood samples were analysed with two dimensional polyacrylamide gel electrophoresis (2D PAGE), MALDI TOF/TOF and nanoLCMS/MS. The milk samples were analysed with MALDI TOF/TOF and nanoLC-MS/MS. Using the optimized gel based proteomic approach,
we have succesfully separated 143 proteins in the group1, 139 proteins in the group2 and 136 proteins in the group3, but we could not find significant differences between groups in the expression pattern. Using MALDI TOF/TOF and nanoLC-IonTrap MS, we have found eleven protein sequences those were expressed only in the samples of good longevity group.
Examination of repeatability of ultrasound scanning technique in young bulls43-48Views:105
The author’s goal with the methodology examination was to determine repeatability of taking and evaluating ultrasound images. In the trial an operator person took two images about the ribeye area and rump fat thickness (P8) of every young bull, which were measured by the same person in four repetitions. Also, we had altogether eight images about the same part of the body. Images were collected using Falco 100 (Pie Medical) real-time ultrasonic scanner equipped with an ASP 3,5 MHz, 18 cm linear array transducer.
Recovery and confirmation of Haemonchus contortus from abomasal contents of roe deer (Capreolus capreolus) in Eastern-Hungary (Biharugra): A diagnostic case study59-62Views:100
Gastrointestinal parasites are ubiquitous. They occur both in wild and domesticated animals. Among such parasites of veterinary importance is the trichostrongyle worms, out of which the Haemonchus contortus species is regarded as the most pathogenic one in the small ruminant industry. The occurrence of this parasite in the sheep flock is now very well documented and an established fact in Europe, although the parasite was original of the warmer climatic region. Studies on the cross-transmission of H. contortus between the wild and domesticated animals are also on the rise although the question of the direction of transmission is still debated. This is an important area that needs to be addressed as it could potentially contribute indirectly to mitigating anthelmintic resistance. Hungary also has reported its share of the occurrence of the parasite, mainly in the sheep flock and a certain population of roe deer. The study presented here is the preliminary results of a diagnostic case study that confirms the presence of H. contortus in wild ruminant deer species that are close to the domesticated sheep population.
The effect of β-glucan, carotenoids, oligosaccharides and anthocyanins on bacteria groups of excreta in broiler chickens15-20Views:168
This study was conducted to examine the effect of natural compounds, such as β-glucan, carotenoids, oligosaccharides, and anthocyanins in the diet on bacteria gropus of excreta in Ross 308 broiler chickens. Chickens were fed 5 diets: control (basal) diet, a diet supplemented by β-glucan at 0.05%, and diets supplemented by carotenoids, oligosaccharides, or anthocyanins at 0.5% of each compound. On experimental day 19, excreta were collected to determine the proportion of Lactobacillus, Bifidobacterium, Campylobacter, Clostridium, and Escherichia coli. Samples were collected aseptically and snap-frozen in liquid nitrogen. Bacterial DNA was isolated from samples, then polymerase chain reaction using primer pairs designed to the 16S rDNA of bacterial groups were applied to define the proportion of the mentioned bacteria. Another universal primer pair was used to amplify a region of 16S rDNA of all the examined bacteria. Proportion of each bacterial groups was determined relatively to the intensity of universal PCR product band by gel documenting system and ImageLab software. Based on the results, carotenoids and anthocyanins increased the proportion of Bifidobacterium, which might imply the beneficial effects of the mentioned compounds on the bacteria composition of excreta.
Effect of selenium supplement on proteome of chicken liver9-13Views:136
The aims of the present study were to optimize a two dimensional polyacrylamide gel electrophoresis method to chicken liver proteome and to determine the changes of protein expression caused by selenium. Twelve broiler chicken were used in this experiment. The selenium intake was 0,2 mg/kg in the control group (6 chicken) and 4,25 mg/kg in the experimental group (6 chicken). Using the optimized proteomic approach, we have succesfully separated 747 proteins in the experimental group and 741 proteins in the control group. We found six proteins, wich expressed only in the samples of experimental group. Further investigations need to determine these six proteins with mass spectrometry (MS) and look for the correlation between the physiological effects of selenium and the expression of these proteins.
Effects of bioactive plant extracts on immune-related gene expression of common carp (Cyprinus carpio)49-56Views:240
In recent years, intensive fish farming has led to an outbreak of several diseases, and the health status of fish can affect the economy of aquaculture. Since fish health and intestinal health are in correlation, it may also have an impact on immunity. Accordingly, many natural feed additives are being used to improve immune functions. In our study, carotenoids, oligosaccharides, and anthocyanins were applied at 1 m/m% in feed to investigate their effects on cytokines, such as interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and interferon regulatory factor-1 (IRF-1) in spleen and mid-intestine of 6 months old carp. Gene expression analysis was carried out to examine IL-1β, IL-8, TNF-α, and IRF-1 mRNA levels in fish spleen and mid-intestine. The gene expression level of pro-inflammatory IL-1β decreased in the mid-intestine of carotenoid-fed carp compared to anthocyanin supplemented group, but the effects of the bioactive plant extracts were not observed on the examined cytokines compared to control fish.