Identification of Differentially Expressed Genes after Endurance Runs in Karbadian Horses to Determine Candidates for Stress Indicators and Performance Capability.

RNA sequencing makes it possible to uncover genetic mechanisms that underlie certain performance traits. In order to gain a deeper insight into the genetic background and biological processes involved in endurance performance in horses, the changes in the gene expression profiles induced by endurance runs over long (70 km) and short (15 km) distances in the blood of Kabardian horses (Equus caballus) were analyzed. For the long-distance runs, we identified 1484 up- and 691 downregulated genes, while after short-distance runs, only 13 up- and 8 downregulated genes (FC > |1.5|; p < 0.05) were found. These differentially expressed genes (DEGs) are involved in processes and pathways that are primarily related to stress response (interleukin production, activation of inflammatory system) but also to metabolism (carbohydrate catabolic process, lipid biosynthesis, NADP metabolic process). The most important genes involved in these processes therefore represent good candidates for the monitoring and evaluation of the performance of horses in order to avoid excessive demands when endurance performance is required, like ACOD1, CCL5, CD40LG, FOS, IL1R2, IL20RA, and IL22RA2, on the one hand, and, on the other hand, for assessing the suitability of a horse for endurance races, like GATA2, GYG1, HIF1A, MOGAT1, PFKFB3, PLIN5, SIK1, and STBD1.

Estimates of Water Consumption of Growing Pigs under Practical Conditions Based on Climate and Performance Data.

The water consumption of fattening pigs was recorded under practical conditions and compared with calculated water consumption. The experiment was carried out in the summer of 2020 with 79 fattening pigs. Data loggers were used to record the climate data, such as temperature and relative humidity. These data were used to calculate the temperature-humidity index (THI). It was found that there were sometimes considerable discrepancies between the measured and the calculated water consumption. One possible reason for this discrepancy could be the age of the existing water requirement equations, as in recent decades there has been a clear breeding development and thus a strong increase in pig performance. Based on these deviations, six new water consumption equations were established, which considered the variables body weight (BW), temperature, THI and feed consumption. It was found that the THI and BW should be included in one equation as predictor variables and the evaluation also showed good results. Its use, in practice, should also be considered. Overall, it became apparent that there is still a need for further research to make water consumption equations more precise. This would require a larger database.

Does the Implementation of an Animal Welfare Programme on a Farm Yield a Demonstrable Improvement in Fattening Pig Welfare?

In the course of social criticism of fattening pig farming, an animal welfare programme called "Initiative Animal Welfare" (ITW) was founded to increase animal welfare in pig farming in Germany. Furthermore, there is a legal obligation to record animal welfare parameters as a self-monitoring measure. The "German Association for Technology and Structures in Agriculture" published a guideline on the applicable animal welfare criteria. This guide formed the basis of this study's data collection. The aim was to apply the animal welfare parameters on farms by comparing the results between farms participating in ITW with those not participating. A cumulative score was calculated by evaluating the collected data. In addition, the relative risk was calculated in order to estimate the risk of finding a negative expression of a parameter. Our data show that ITW farms did not perform significantly better than the farms without ITW in terms of both the cumulative score and the relative risk. Overall, it must be considered that in both farm variants the occurrence of negative evaluations was very rare and the visited farms thus certainly can be considered to be well-managed farms. Climate parameters were recorded in each compartment and showed no significant differences in most cases.

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis.

BACKGROUND: In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. METHODS: To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. RESULTS: We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. CONCLUSIONS: ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial.

The Effect of Algae or Insect Supplementation as Alternative Protein Sources on the Volatile Profile of Chicken Meat.

The aim of this study was to investigate the differences in the volatile profile of meat from chickens fed with alternative protein diets (such as algae or insect) through two different trials. In Trial 1, broiler chicken at one day of age were randomly allocated to three experimental groups: a basal control diet (C) and two groups in which the soybean meal was replaced at 75% (in the starter phase) and 50% (in the grower phase) with partially defatted Hermetia illucens (HI) larvae or Arthrospira platensis (SP). In Trial 2, broiler chickens were housed and reared similar to Trial 1, with the exception that the experimental diets replaced soybean meal with either 100% partially defatted HI or 100% SP. In both trials, chickens were slaughtered at day 35. Per group, 10 chickens were submitted to volatile analysis by using solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) analysis. Results in both trials showed that levels of several lipid-derived compounds were found to be lower in chickens fed an HI diet, which could be linked to a possibly lower level of polyunsaturated fatty acid content in HI-fed chicken. In addition, the dietary treatments could be discriminated based on the volatile profile, i.e., the substitution of soy with HI or SP distinctively affected the levels of flavor compounds.

Survey data to identify the selection criteria used by breeders of four strains of Pakistani beetal goats.

This article presents raw data from a survey conducted to identify the selection criteria of breeders raising either of four strains of Beetal goats, namely Beetal Faisalabadi, Beetal Makhi-Cheeni, Beetal Nuqri, and Beetal Rahim Yar Khan. After a pre-survey, a questionnaire was developed and a survey was conducted at four sites of the Punjab province of Pakistan: Faisalabad/Sahiwal, Bahawalpur/Bahawalnagar, Rajanpur, and Rahim Yar Khan. Each of these sites was the home tract of one strain. During the survey breeders (n = 162) were asked to rank the traits of their selection criteria based on the relative importance of those traits. Furthermore, the prevailing production system was also characterized by the breeders. For the interpretation of the results of this survey the readers are referred to Ref. [1]. The raw data set provided in this article can be extended in the future to include more strains of Beetal goats as well as other goat breeds. The selection criteria of breeders can change over time. This data set can also be used in future studies to investigate the temporal changes in the relative importance of different traits for the breeders. The factors potentially influencing those changes can also be investigated. This data set can further be utilized to design community based breeding plans tailored to the needs of the goat farming community.

Acceptance and Feasibility of a Guideline for the Animal Welfare Assessment of Fattening Pigs from Farmers' Point of View.

The welfare of farm animals is being increasingly discussed in society and politics. To evaluate animal welfare, indicator systems are often used. Such a system has been developed by the German Association for Technology and Structures in Agriculture and suggested in the publication "Animal Welfare Indicators: Practical Guide-Pigs". The association's aim is to provide farmers with a useful method for recording the welfare of pigs. Crucial for the acceptance of the guide by farmers is a high degree of feasibility of the recommended indicators as well as the proposed methods for their recording. To evaluate this, 40 farmers keeping fattening pigs were interviewed. The guided semi-structured interview was conducted on the farms after the farmers evaluated the welfare of their fattening pigs according to the guide. The results are: Apart from the indicators faecal soiling and tail length, all the other eleven indicators are accepted for the assessment of fattening pig welfare by a majority of the interviewed farmers (between 57.5% and 90% acceptance per indicator). Furthermore, the feasibility of the individual indicators was assessed as being positive. The relationship between time expenditure and benefit was rated on a five-point scale at an average of 3.1 (medium), which clearly shows that there is a need for further development of this guide. Some possible changes with a potential for improvement could be identified; for example, the aggregation of the results after the collection of the individual indicators to an overall result that can be compared and interpreted.

Animal Welfare Assessment of Fattening Pigs: A Case Study on Sample Validity.

A guide for animal welfare assessment of fattening pigs recommends recording some of the indicators for a sample of the animals from a herd. However, it is not certain whether the herd's level of welfare can be correctly judged using a random sample. Therefore, both the true prevalences of welfare indicators in a full census and the estimated prevalences of the indicators based upon simulated samples taken according to five strategies (termed S1 to S5) were determined. Deviations from the true level of animal welfare in the herd due to the sampling were recorded and analyzed. Depending on the strategy, between 12% and 43% of the samples over- or underestimated the true prevalences by more than 50%. The validity of the sampling strategies was evaluated using the normalized root-mean-squared error (NRMSE) and the relative bias (RB). In terms of accuracy, the strategies differed only slightly (between NRMSE = 0.13 for S2 and NRMSE = 0.19 for S4). However, the strategies varied more obviously regarding the bias (between RB = -0.0002 for S1 and RB = -0.0370 for S5). The described results are the outcome of an initial case study on the sample validity of the indicators and have to be verified using the data of more herds.

Identification of Candidate Signature Genes and Key Regulators Associated With Trypanotolerance in the Sheko Breed.

African animal trypanosomiasis (AAT) is caused by a protozoan parasite that affects the health of livestock. Livestock production in Ethiopia is severely hampered by AAT and various controlling measures were not successful to eradicate the disease. AAT affects the indigenous breeds in varying degrees. However, the Sheko breed shows better trypanotolerance than other breeds. The tolerance attributes of Sheko are believed to be associated with its taurine genetic background but the genetic controls of these tolerance attributes of Sheko are not well understood. In order to investigate the level of taurine background in the genome, we compare the genome of Sheko with that of 11 other African breeds. We find that Sheko has an admixed genome composed of taurine and indicine ancestries. We apply three methods: (i) The integrated haplotype score (iHS), (ii) the standardized log ratio of integrated site specific extended haplotype homozygosity between populations (Rsb), and (iii) the composite likelihood ratio (CLR) method to discover selective sweeps in the Sheko genome. We identify 99 genomic regions harboring 364 signature genes in Sheko. Out of the signature genes, 15 genes are selected based on their biological importance described in the literature. We also identify 13 overrepresented pathways and 10 master regulators in Sheko using the TRANSPATH database in the geneXplain platform. Most of the pathways are related with oxidative stress responses indicating a possible selection response against the induction of oxidative stress following trypanosomiasis infection in Sheko. Furthermore, we present for the first time the importance of master regulators involved in trypanotolerance not only for the Sheko breed but also in the context of cattle genomics. Our finding shows that the master regulator Caspase is a key protease which plays a major role for the emergence of adaptive immunity in harmony with the other master regulators. These results suggest that designing and implementing genetic intervention strategies is necessary to improve the performance of susceptible animals. Moreover, the master regulatory analysis suggests potential candidate therapeutic targets for the development of new drugs for trypanosomiasis treatment.

Computational identification of tissue-specific transcription factor cooperation in ten cattle tissues.

Transcription factors (TFs) are a special class of DNA-binding proteins that orchestrate gene transcription by recruiting other TFs, co-activators or co-repressors. Their combinatorial interplay in higher organisms maintains homeostasis and governs cell identity by finely controlling and regulating tissue-specific gene expression. Despite the rich literature on the importance of cooperative TFs for deciphering the mechanisms of individual regulatory programs that control tissue specificity in several organisms such as human, mouse, or Drosophila melanogaster, to date, there is still need for a comprehensive study to detect specific TF cooperations in regulatory processes of cattle tissues. To address the needs of knowledge about specific combinatorial gene regulation in cattle tissues, we made use of three publicly available RNA-seq datasets and obtained tissue-specific gene (TSG) sets for ten tissues (heart, lung, liver, kidney, duodenum, muscle tissue, adipose tissue, colon, spleen and testis). By analyzing these TSG-sets, tissue-specific TF cooperations of each tissue have been identified. The results reveal that similar to the combinatorial regulatory events of model organisms, TFs change their partners depending on their biological functions in different tissues. Particularly with regard to preferential partner choice of the transcription factors STAT3 and NR2C2, this phenomenon has been highlighted with their five different specific cooperation partners in multiple tissues. The information about cooperative TFs could be promising: i) to understand the molecular mechanisms of regulating processes; and ii) to extend the existing knowledge on the importance of single TFs in cattle tissues.

Multi-Approach Analysis for the Identification of Proteases within Birch Pollen.

Birch pollen allergy is highly prevalent, with up to 100 million reported cases worldwide. Proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. Until now the protease content of Betula verrucosa, a birch species endemic to the northern hemisphere has not been studied in detail. Hence, we aim to identify and characterise pollen and bacteria-derived proteases found within birch pollen. The pollen transcriptome was constructed via de novo transcriptome sequencing and analysis of the proteome was achieved via mass spectrometry; a cross-comparison of the two databases was then performed. A total of 42 individual proteases were identified at the proteomic level. Further clustering of proteases into their distinct catalytic classes revealed serine, cysteine, aspartic, threonine, and metallo-proteases. Further to this, protease activity of the pollen was quantified using a fluorescently-labelled casein substrate protease assay, as 0.61 ng/mg of pollen. A large number of bacterial strains were isolated from freshly collected birch pollen and zymographic gels with gelatinase and casein, enabled visualisation of proteolytic activity of the pollen and the collected bacterial strains. We report the successful discovery of pollen and bacteria-derived proteases of Betula verrucosa.

Identification of Proteases and Protease Inhibitors in Allergenic and Non-Allergenic Pollen.

Pollen is one of the most common causes of allergy worldwide, making the study of their molecular composition crucial for the advancement of allergy research. Despite substantial efforts in this field, it is not yet clear why some plant pollens strongly provoke allergies while others do not. However, proteases and protease inhibitors from allergen sources are known to play an important role in the development of pollen allergies. In this study, we aim to uncover differences in the transcriptional pattern of proteases and protease inhibitors in Betula verrucosa and Pinus sylvestris pollen as models for high and low allergenic potential, respectively. We applied RNA sequencing to Betula verrucosa and Pinus sylvestris pollen. After de-novo assembly we derived general functional profiles of the protein coding transcripts. By utilization of domain based functional annotation we identified potential proteases and protease inhibitors and compared their expression in the two types of pollen. Functional profiles are highly similar between Betula verrucosa and Pinus sylvestris pollen. Both pollen contain proteases and inhibitors from 53 and 7 Pfam families, respectively. Some of the members comprised within those families are implicated in facilitating allergen entry, while others are known allergens themselves. Our work revealed several candidate proteins which, with further investigation, represent exciting new leads in elucidating the process behind allergic sensitization.

The crystal structure of Erwinia amylovora AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions.

AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.

A comparison of the functional traits of common reed (Phragmites australis) in northern China: aquatic vs. terrestrial ecotypes.

Common reed (Phragmites australis (Cav.) Trin. ex Steud.) is distributed widely throughout the world with various ecotypes. This research compares the functional traits and biomass allocation patterns of two contrasting reed ecotypes. Twelve pairs of aquatic and terrestrial reed samples were collected in northern China. Significant differences in functional traits between the two reed ecotypes were observed, while biomass allocation patterns of reed organs did not differ significantly except for at the root. The dry matter content (DMC) in the whole of the reed plant, leaf, root, and rhizome was higher; while the specific leaf area (SLA) and specific root length (SRL) were lower in terrestrial versus aquatic reed. The biomass allocation in organs of the two forms of reed was isometric, only root in the terrestrial habitat increased faster with an increase in the whole plant biomass. It can be affirmed that aquatic and terrestrial reed that adapt to different environments generally has distinct leaf and root functional traits but isometric biomass allocation patterns. This suggests different resource acquisition strategies: (1) aquatic reed grows faster with high SLA and SRL and is more responsive to the environment, while (2) terrestrial reed with high DMC grows slower and is less responsive to the adverse environment (e.g. dry soil conditions).

Equivalent indels--ambiguous functional classes and redundancy in databases.

There is considerable interest in studying sequenced variations. However, while the positions of substitutions are uniquely identifiable by sequence alignment, the location of insertions and deletions still poses problems. Each insertion and deletion causes a change of sequence. Yet, due to low complexity or repetitive sequence structures, the same indel can sometimes be annotated in different ways. Two indels which differ in allele sequence and position can be one and the same, i.e. the alternative sequence of the whole chromosome is identical in both cases and, therefore, the two deletions are biologically equivalent. In such a case, it is impossible to identify the exact position of an indel merely based on sequence alignment. Thus, variation entries in a mutation database are not necessarily uniquely defined. We prove the existence of a contiguous region around an indel in which all deletions of the same length are biologically identical. Databases often show only one of several possible locations for a given variation. Furthermore, different data base entries can represent equivalent variation events. We identified 1,045,590 such problematic entries of insertions and deletions out of 5,860,408 indel entries in the current human database of Ensembl. Equivalent indels are found in sequence regions of different functions like exons, introns or 5' and 3' UTRs. One and the same variation can be assigned to several different functional classifications of which only one is correct. We implemented an algorithm that determines for each indel database entry its complete set of equivalent indels which is uniquely characterized by the indel itself and a given interval of the reference sequence.

Effect of the myostatin locus on muscle mass and intramuscular fat content in a cross between mouse lines selected for hypermuscularity.

BACKGROUND: This study is aimed at the analysis of genetic and physiological effects of myostatin on economically relevant meat quality traits in a genetic background of high muscularity. For this purpose, we generated G(3) populations of reciprocal crosses between the two hypermuscular mouse lines BMMI866, which carries a myostatin mutation and is lean, and BMMI806, which has high intramuscular and body fat content. To assess the relationship between muscle mass, body composition and muscle quality traits, we also analysed intramuscular fat content (IMF), water holding capacity (WHC), and additional physiological parameters in M. quadriceps and M. longissimus in 308 G(3)-animals. RESULTS: We found that individuals with larger muscles have significantly lower total body fat (r = -0.28) and IMF (r = -0.64), and in females, a lower WHC (r = -0.35). In males, higher muscle mass was also significantly correlated with higher glycogen contents (r = 0.2) and lower carcass pH-values 24 hours after dissection (r = -0.19). Linkage analyses confirmed the influence of the myostatin mutation on higher lean mass (1.35 g), reduced body fat content (-1.15%), and lower IMF in M. longissimus (-0.13%) and M. quadriceps (-0.07%). No effect was found for WHC. A large proportion of variation of intramuscular fat content of the M. longissimus at the myostatin locus could be explained by sex (23%) and direction-of-cross effects (26%). The effects were higher in males (+0.41%). An additional locus with negative over-dominance effects on total fat mass (-0.55 g) was identified on chromosome 16 at 94 Mb (86-94 Mb) which concurs with fat related QTL in syntenic regions on SSC13 in pigs and BTA1 in cattle. CONCLUSION: The data shows QTL effects on mouse muscle that are similar to those previously observed in livestock, supporting the mouse model. New information from the mouse model helps to describe variation in meat quantity and quality, and thus contribute to research in livestock.

Correction: Equivalent Indels - Ambiguous Functional Classes and Redundancy in Databases.

[This corrects the article DOI: 10.1371/journal.pone.0062803.].

Growth of influenza A virus is not impeded by simultaneous removal of the cholesterol-binding and acylation sites in the M2 protein.

Influenza virus assembly and budding occur in the 'budozone', a coalesced raft domain in the plasma membrane. The viral transmembrane protein M2 is implicated in virus particle scission, the ultimate step in virus budding, probably by wedge-like insertion of an amphiphilic helix into the membrane. In order to do this, M2 is hypothesized to be targeted to the edge of the budozone, mediated by acylation and cholesterol binding. It was recently shown that acylation and cholesterol binding affect the membrane association of the cytoplasmic tail of M2 and targeting of the protein to coalesced rafts. This study tested whether combined removal of the acylation site (C50) and the cholesterol recognition/interaction amino acid consensus motifs (key residues Y52 and Y57) in the amphiphilic helix of M2 influenced virus formation. Recombinant influenza viruses were generated in the influenza strain A/WSN/33 background with mutations in one or both of these features. In comparison with the wild-type, all mutant viruses showed very similar growth kinetics in various cell types. Wild-type and mutant viruses differed in their relative M2 content but not regarding the major structural proteins. The morphology of the viruses was not affected by mutating M2. Moreover, wild-type and mutant viruses showed comparable competitive fitness in infected cells. Lastly, a global comparison of M2 sequences revealed that there are natural virus strains with M2 devoid of both lipid-association motifs. Taken together, these results indicate that the acylation and cholesterol-binding motifs in M2 are not crucial for the replication of influenza virus in cell culture, indicating that other factors can target M2 to the budding site.

NovelSNPer: A Fast Tool for the Identification and Characterization of Novel SNPs and InDels.

Typically, next-generation resequencing projects produce large lists of variants. NovelSNPer is a software tool that permits fast and efficient processing of such output lists. In a first step, NovelSNPer determines if a variant represents a known variant or a previously unknown variant. In a second step, each variant is classified into one of 15 SNP classes or 19 InDel classes. Beside the classes used by Ensembl, we introduce POTENTIAL_START_GAINED and START_LOST as new functional classes and present a classification scheme for InDels. NovelSNPer is based upon the gene structure information stored in Ensembl. It processes two million SNPs in six hours. The tool can be used online or downloaded.

Genetic determinants for intramuscular fat content and water-holding capacity in mice selected for high muscle mass.

Intramuscular fat content and water-holding capacity are important traits in livestock as they influence meat quality, nutritive value of the muscle, and animal health. As a model for livestock, two inbred lines of the Berlin Muscle Mouse population, which had been long-term selected for high muscle mass, were used to identify genomic regions affecting intramuscular fat content and water-holding capacity. The intramuscular fat content of the Musculus longissimus was on average 1.4 times higher in BMMI806 than in BMMI816 mice. This was accompanied by a 1.5 times lower water-holding capacity of the Musculus quadriceps in BMMI816 mice. Linkage analyses with 332 G(3) animals of reciprocal crosses between these two lines revealed quantitative trait loci for intramuscular fat content on chromosome 7 and for water-holding capacity on chromosome 2. In part, the identified loci coincide with syntenic regions in pigs in which genetic effects for the same traits were found. Therefore, these muscle-weight-selected mouse lines and the produced intercross populations are valuable genetic resources to identify genes that could also contribute to meat quality in other species.