Investigation of candidate genes for metabolic disorders expressed in liver and pituitary gland by comparing the RNA-seq data of Polish-HF and Polish-Red cattle

Background: Metabolic disorder is a major health problem in dairy cattle, particularly to high milk producing dairy cattle. It is worthily emphasized that metabolic diseases have a very complex etiology and pathogenesis, and the impact of these diseases on hepatic and pituitary gland gene expression and organism oxidative balance is not fully described. The presented study was aimed to determine and predict the hepatic and pituitary gland expression of potential candidate genes in context to maintenance of oxidative balance, negative nitrogen balance, as well as ketosis in Polish HF and Polish Red cattle.Methods: Based on the RNA-seq experimental data, we investigated the candidate genes (SOD1, SOD2, SOD3, GPx2, GPx3, GPx5, GPx6, GPx7, GPx8, BDH1, FN1, ACSL3, HMGCL, HMGCS2, BDH2, ACSL6, ACAT2, IDH3B, ACAT1, HMGCS1, ACSL4, ACSL1, PC, CPT1A, OXCT1 and ACSL5 respectively) expressions in liver and pituitary gland tissues of Polish HF and Polish Red cattle. The RNA-seq experimental design comprised of young bulls aged between 6 to 12 months were investigated. For each breed, six liver and six pituitary gland tissues were sequenced using Next-seq 500 illumina platform. The RNA-seq expression data were normalized by the reads per kilobase of exon per million reads mapped (RPKM) method.Results: By comparing the RNA-seq data of liver and pituitary gland tissues, the investigated candidate genes were highly expressed in the hepatic tissues than to pituitary gland in investigated cattle breeds. However, by comparing the Polish HF and Polish Red cattle breeds, results revealed a similar trend of gene expression profiling of all investigated candidate genes for both metabolic tissues. In case of hepatic gene expression profiling, the SOD1, FN1, HMGCL, HMGCS2, ACAT2, ACAT1, HMGCS1, ACSL1 and ACSL5 were highly expressed (FPKM values of >40), followed by SOD2, GPX3, IDH3B, PC and BDH2 as moderately expressed (FPKM values: >10 to <40), and averagely expressed SOD3, GPX5, GPX6, GPX7, GPX2, GPX8, BDH1, ACSL3, ACSL6, ACSL4, CPT1A and OXCT1 respectively, in Polish HF and Polish Red breeds. In case of pituitary gland gene expression profiling, the SOD1 and GPx3 were highly expressed (FPKM values of >40), followed by SOD2, GPX8, IDH3B, ACAT1, ACSL4 and PC as moderately expressed (FPKM values: >10 to <40), and averagely expressed SOD3, GPX3,GPX5, GPX6, GPX7, GPX2, BDH1, BDH2, ACSL3, ACSL6, CPT1A, OXCT1, FN1, HMGCL, HMGCS2, ACAT2, ACAT1, HMGCS1, ACSL1 and ACSL5 respectively, in Polish HF and Polish Red breeds.Conclusions: Based on this presented results on hepatic and pituitary gland gene expression, a further research plan is an essential pre-requisite to validate the identified candidate genes. Study indicated the understanding the genetic factors that predispose metabolic disorders in cattle would benefit the dairy industry as a whole by providing producers, breeding services, and veterinarians a tool to forecast a cow’s susceptibility to metabolic disorders

Examination of the redox status of calves during the milk feeding period in a Hungarian large-scale dairy farm

During the milk feeding period (from birth to weaning, generally for 60-70 days), calves receive feed milk or milk replacer rich in both protein and energy. Young animals show intense physical development and growth. Intensive oxidative metabolic processes, inadequate antioxidant defense system, oxidative stress can develop, which adversely affects the health and productivity of calves due to its cell-damaging effects. This justifies continuous monitoring of the redox status of the animals during the calf rearing period for early detection of oxidative stress. This may provide a basis for targeted antioxidant treatments to reduce calf disease-related losses

Identifikacija monomorfnih i polimorfnih gena povezanih s recesivno uvjetovanim poremećajima plodnosti holštajnskih krava u Kazahstanu

Haplotypes of candidate genes namely: apoptotic protease activating factor 1 (APAF1 p.Q579X or HH1), glycinamide ribonucleotide formyltransferase (GART or HH4), structural maintenance of chromosomes 2 (SMC2 or HH3), and haplotype cholesterol deficiency (HCD) genes associated with recessive fertility defects (loss of fertility) were investigated in imported Canadian Holstein cows reared at “Medeu Commerce” LLP breeding farm in Kazakhstan. The genotypic profiling of the APAF1/HH1, GART/HH4 fertility haplotype carriers was carried out by PCR-RFLP methods using BstC8I and Tru9I and MseI, while the genotypic profiling of the SMC2/HH3, and HCD fertility haplotype carriers was carried out using our own primer designed by internal primer marker methods. The study revealed that the PCR-RFLP diagnostic markers APAF1/HH1 and GART/HH4 for recessive fertility defects were monomorphic in the Canadian Holstein cows investigated. However, the diagnostic markers SMC2/HH3 and HCD fertility haplotype carriers (our own design diagnostic markers) were polymorphic, with frequencies of 3% and 11%, respectively, in the investigated Canadian Holstein cows. The study concluded that genetic monitoring of recessive fertility defects enables the timely identification of carriers of harmful lethal mutations, and control of the fertility haplotype elimination process.U krava holštajnske pasmine, uvezenih iz Kanade i uzgojenih na poljoprivrednom gospodarstvu Medeu Commerce LLP u Kazahstanu, istraživani su haplotipovi slijedećih kandidatnih gena za koje se smatra da su povezani s poremećajima (gubitkom) plodnosti: faktor koji aktivira apoptotsku proteazu 1 (APAF1 p.Q579X ili HH1), glicinamidribonukleotidna formiltransferaza (GART ili HH4), strukturno održavanje kromosoma 2 (SMC2 ili HH3) i haplotip za manjak kolesterola (HCD). Genotipsko profiliranje nositelja haplotipova APAF1/HH1, GART/HH4 provedeno je metodom PCR-RFLP uz upotrebu BstC8I i Tru9I te MseI, dok je genotipsko profiliranje nositelja haplotipova SMC2/ HH3, i HCD provedeno vlastitim primerima, oblikovanim internim metodama. Rezultati su pokazali da su metodom PCR-RFLP dijagnostički markeri APAF1/HH1 i GART/HH4 za recesivne poremećaje plodnosti u istraživanih krava bili monomorfni. Istovremeno, dijagnostički markeri nositelja haplotipova SMC2/HH3 i HCD (oblikovani prema vlastitim metodama) bili su polimorfni, s učestalošću od 3 % i 11 %. Zaključeno je da genetski nadzor recesivnih poremećaja plodnosti omogućuje pravodobno identificiranje nositelja štetnih letalnih mutacija i kontrolu eliminacije haplotipova koji štetno utječu na plodnost

Effect of a single, oral administration of selenitetriglycerides, at two dose rates, on blood selenium status and haematological and biochemical parameters in Holstein-Friesian calves

peer-reviewedBackground Selenitetriglycerides are biologically active, organic forms of selenium formed as a result of the modification of selenic acid and sunflower oil. Studies in rats have shown that they are well absorbed and of low toxicity. There are no published studies on selenitetriglycerides supplementation in calves. Results In this study, selenitetriglycerides were administered once orally on the 2nd day of life at a dose of 0.5 or 1 mg Se/kg body weight to each of six Holstein-Friesian calves while six control calves were not supplemented. Blood for determination of selenium concentration, glutathione peroxidase activity, haematological parameters, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase activities and glucose, total protein, albumin, triglycerides, cholesterol, urea, and creatinine concentration was collected before supplementation (day 0) and 1, 2, 5, 10 and 14 days after supplementation. Selenitetriglycerides administration increased (P < 0.01) serum selenium concentration in supplemented calves as early as day1, from a mean of 63.4 to 184.22 µg/l in calves receiving selenium at a dose of 0.5 mg/kg BW, and from 63.17 to 200.33 µg/l in calves receiving 1 mg/kg. Serum selenium concentrations remained significantly higher compared to the control group throughout the experiment. Glutathione peroxidase activity was higher in supplemented than control calves, significantly so in animals receiving the 1 mg/kg dose of Se on the 10th and 14th days (P < 0.05). There were no significant differences in the haematological and biochemical parameters between the groups. Conclusions This experiment showed that supplementation with selenitetriglycerides could significantly improve blood selenium status in calves without adverse effects on haematological or biochemical parameters. These findings are essential prerequisites for future studies on selenitetriglycerides supplementation to manage clinical selenium deficiency in calves

Multiclass method to determine emerging pollutants in bats using a non-invasive approach based on guano matrix

Emerging pollutants have been ubiquitously found in environmental compartments, while there is scarce information about these substances and their effects on health status in wild terrestrial mammals. Bat species are very sensitive animals to any changes in the environment and are considered one of the best bioindicators of the quality of the environment to terrestrial wildlife. To acquire a better knowledge of the environmental exposure to these animals, a multiclass method is proposed to determine 20 emerging pollutants (six perfluoroalkyl substances (PFAS), four parabens (PB), four benzophenones (BP), a plasticizer (BPA), and five surfactants (four linear alkylbenzene sulfonates (LAS) and nonylphenol (NP)) in bats using a non-invasive approach based on guano matrix. Sample treatment involved ultrasonic solvent extraction and dispersive solid phase extraction prior to analysis in a single run with liquid chromatography-tandem mass spectrometry. The main variables affecting the extraction and clean-up steps were evaluated using single and multivariate strategies. Under the optimized conditions, satisfactory analytical characteristics in terms of linearity, recoveries (>80 %), precision (RSD < 24 %) and method quantification limits (from 0.01 to 64 ng/g dry weight) were obtained. Furthermore, and as a proof of concept, guano samples from a bat reference population were collected from a colony located in Brenna village, in south Poland. The results confirm the exposure of wild bats to emerging pollutants (LAS, PFAS, and PB compounds were frequently detected in the samples) and the suitability of the bat guano matrix for understanding the environmental exposure in terrestrial mammals

Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology

BackgroundRNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. ResultsThe RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive QTL/CG analysis of 110 QTL/CG with RNA-seq data identified 20 monomorphic SNP hit loci (CARTPT, GAD1, GDF5, GHRH, GHRL, GRB10, IGFBPL1, IGFL1, LEP, LHX4, MC4R, MSTN, NKAIN1, PLAG1, POU1F1, SDR16C5, SH2B2, TOX, UCP3 and WNT10B) in all three cattle breeds. However, six SNP loci (CCSER1, GHR, KCNIP4, MTSS1, EGFR and NSMCE2) were identified as highly polymorphic among the cattle breeds. ConclusionsThis study identified breed-specific SNPs with greater SNP ratio and excellent mapping coverage, as well as monomorphic and highly polymorphic putative SNP loci within QTL/CGs of bovine liver tissue. A breed-specific SNP-db constructed for bovine liver yielded nearly six million SNPs. In addition, a KASPTM SNP genotyping assay, as a reliable cost-effective method, successfully validated the breed-specific putative SNPs originating from the RNA-seq experiments

Promjene u sustavu grušanja krvi u junica holštajnsko-frizijske pasmine u tijeku klamidiozeb

The objective of this study was to describe changes in the coagulation and fibrinolytic systems of Holstein- Friesian (H-F) heifers infected naturally with Chlamydia spp. in Kazakhstan. Blood coagulation and morphology tests were performed in 30 heifers, including 20 infected animals (experimental group I) and 10 clinically healthy and uninfected animals (control group II). In the laboratory tests, the following blood parameters were determined: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), antithrombin III activity (ATIII), fibrinogen (FBG), D-dimer (D-D) concentrations, thrombocyte counts (PLT) and leukocyte counts (WBC). Decreased PT and APTT values in the blood plasma of heifers infected with Chlamydia spp. were indicative of intensified procoagulant activity in such an acute infection. Increased ATIII activity could point to the activation of the anticoagulant system to prevent excessive fibrin production in diseased animals. The results indicate that the hemostatic system was activated in heifers with symptoms of chlamydiosis, and regulatory mechanisms prevented the development of life-threatening conditions such as thrombosis or disseminated intravascular coagulation (DIC).ŚőCilj je ovog istraživanja bio opisati promjene u koagulacijskom i fibrinolitičkom sustavu holštajnskofrizijske pasmine junica prirodno zaraženih vrstama roda Chlamydia spp. u Kazakhstanu. Testovi grušanja krvi i morfološki testovi provedeni su na 30 junica od čega 20 zaraženih (pokusna skupina) i 10 klinički zdravih nezaraženih životinja (kontrolna skupina). Uporabom laboratorijskih testova određeni su sljedeći krvni pokazatelji: protrombinsko vrijeme, aktivirano parcijalno tromboplastinsko vrijeme, trombinsko vrijeme, aktivnost antitrombina III, fibrinogen, koncentracije D-dimera (D-D), broj trombocita i broj leukocita. Smanjene vrijednosti protrombinskog vremena i aktiviranog parcijalnog tromboplastinskog vremena junica zaraženih vrstama Chlamydia spp. bile su indikacija za pojačano prokoagulacijsku aktivnost kod takve akutne infekcije. Povećana aktivnost antitrombina III mogla bi upućivati na aktivaciju antikoagulacijskog sustava da bi se spriječila prevelika proizvodnja fibrina u oboljelih životinja. Rezultati upućuju na zaključak da je hemostazni sustav bio aktiviran u junica sa znakovima klamidioze te da su regulacijski mehanizmi spriječili razvoj po život opasnih stanja kao što je tromboza ili diseminirana intravaskularna koagulacija

Epidemiological characteristics and financial losses due to avian aspergillosis in households in the Almaty region, Republic of Kazakhstan

Aspergillosis is a severe fungal disease that affects all species and ages of poultry and leads to significant economic losses within the poultry industry. The economic significance of aspergillosis is associated with direct losses due to poultry mortality, a decline in the production of meat and eggs, feed conversion, and poor growth of recovering poultry. Although a decrease in the production of poultry meat and eggs in Kazakhstan due to this fungal disease has been widely reported, studies on the consequent financial losses on affected farms (households) have not been carried out. This study aimed to estimate the financial losses and epidemiological parameters of avian aspergillosis among households affected by the disease in the Almaty region. To achieve the objectives of the research, a survey was conducted involving affected households from February 2018 to July 2019. The affected poultry were diagnosed based on clinical, macroscopical, and microscopical procedures, and once the infection was confirmed, household owners were interviewed. Data were collected from 183 household owners. The median incidence risk and fatality rates were 39 and 26% in chickens, 42 and 22% in turkeys, and 37 and 33% in geese, respectively, with young poultry having a higher incidence risk and fatality rate than adults. Approximately 92.4% of the household owners treated the affected poultry using natural folk methods and 7.6% of household owners used antifungal drugs and antibiotics, spending a median of US $35.20 (min US$0; max US $400) per household throughout the course of the infection. Egg production was reduced by a median of 58.3$198.50 (min US $11; max US$1,269). The majority of household owners (65%) did not replace their poultry, 9.8% of household owners replaced all their poultry, and the remaining 25.1% replaced only a proportion of the poultry lost at the time of the study. Newly acquired poultry were purchased from neighbors (10.9%), fellow villagers (50%), and state poultry farms (39.1%). This study demonstrates that aspergillosis has an immediate impact on subsistence household owners' livelihoods in the Almaty region of Kazakhstan

Quality control assessment of the RNA-Seq data generated from liver and pituitary transcriptome of Hereford bulls using StrandNGS software

Background: Quality control (QC) assessment is the most critical step in the high-throughput RNA-seq data analysis to characterize the in-depth understanding of genome and transcriptome assembling to a given reference genome. It provides not only a quick insight into the RNA-seq data quality to allow early identification of good or bad RNA-seq data samples, but also to verify the alignment QC checks for further essential high-throughput bioinformatics analysis such as, identification of novel genetic variants, differentially expressed genes (DEGs), gene network and metabolic pathways.Method: After isolation of total RNA from liver (n=15) and pituitary gland (n=15) tissues of young Hereford bulls, the pooled total RNA (n=30) were fragmented using GeneRead rRNA depletion kit (Qiagen, Hilden, Germany) and cDNA library preparation were preformed using ScriptSeqTM v2 RNA-Seq library preparation kit (Epicentre, illumina, USA), followed by high-throughput sequencing of combined liver and pituitary transcriptome using MiSeq reagent kit v2 (illumina, USA) to obtain high quality of paired-end RNA-seq reads of 251 base-pairs (bps). In this paper, the QC assessment of obtained RNA-seq raw data as well as post-alignment QC of processed RNA-seq data of combined liver and pituitary transcriptome (n=30) of Hereford bulls were performed using the strand NGS software v1.3 (Agilent; http://www.strand-ngs.com/) data analysis package. The reads were aligned with Bowtie using default settings against both Bull and Cow genome assembly.Results: Using two runs of MiSeq platform, a total of over 60 million paired-end RNA-seq reads were successfully obtained and submitted to NCBI SRA resources (https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&amp;from_uid=312148). Library complexity plot results revealed 72.02% of duplicate reads with a low library complexity value of 0.28. The pre-alignment QC analysis of raw RNA-seq data revealed the sequence read lengths ranged from 35-251 bp size with more than 50% of all reads with length over 200bp and 10% of reads below 100bp.Conclusion: By testing the RNA-seq methodology on Illumina platform, two MiSeq sequencing runs yielded significantly high quality of 30 million sequencing reads per single MiSeq run. Our initial pre-alignment and post-alignment analysis of RNA-seq data analysis revealed that mapping of the Hereford liver and pituitary gland transcriptome to reference Bos taurus genome was successfully performed, however, more than 50% of all reads with length over 200bp were recovered. Therefore, obtained results concludes that liver and pituitary transcriptome sequencing with rRNA depletion method is less effective than mRNA RNA-seq method

Utjecaj različitih koncentracija mješavine ljuske stijene i bentonita u hrani na rast, pokazatelje u krvi i mesu u afričkog crnog noja (Struthio camelus) iz jugoistočnog Kazahstana

Forty-eight 3-month old African ostrich females were divided into four groups: control and 3 experimental comprising 12 animals each. All the animals were fed the adopted diet and the experimental groups additionally received a feed supplement based on shell rock and bentonite, at a rate of 5 g/kg, 10 g/kg and 15 g/kg, respectively, for 9 months. At the beginning and the end of the experiment blood samples were collected for hematological and biochemical analyses. At the end of the experiment, 3 ostriches from each group were slaughtered and the quality of their meat was examined. At 12 months of age the weight of the birds in the experimental groups was significantly higher (P≤0.05) in comparison to the control group, by 11 kg, 13.3 kg or 16.4 kg, respectively. During the experiment, an increase in the number of red blood cells, leukocytes and hemoglobin was recorded, however all tested hematological parameters remained within the range of physiological values. The biochemical blood parameters in all groups of ostriches did not change significantly at the beginning and the end of experiment. However, the levels of total protein, glucose, Ca and P in the experimental groups showed an upward trend in correlation with the dosage of the tested feed additive. Feed additive contributed to a change in the chemical composition and biological value of the meat. In comparison with the control group, an increase in ash, fat, protein and energy content, the level of mineral elements and vitamins in the meat of the experimental ostriches was noted, in a reliable dependence on the inclusion rate of the feed additive (P≤0.05). We concluded that the use of feed additive positively affects body mass and hematological blood parameters, as well as the nutritional and biological value of ostrich meat.Četrdeset i osam crnih afričkih nojeva, ženki u dobi od tri mjeseca podijeljeno je u četiri skupine. U kontrolnoj i tri pokusne skupine bilo je po 12 životinja. Kontrolna (ili prva) skupina hranjena je prilagođenim obrokom, dok su pokusne skupine, druga, treća i četvrta, dodatno primale dodatak hrani, mješavinu ljuski stijena i bentonita u dozama od 5 g/kg, 10 g/kg ili 15 g/kg tijekom 9 mjeseci. Na početku i na kraju pokusa uzimani su uzorci krvi za hematološke i biokemijske analize. Na kraju pokusa po 3 noja iz svake skupine je žrtvovano te je pretražena kakvoća njihova mesa. U dobi od 12 mjeseci u svim je skupinama utvrđeno značajno povećanje tjelesne mase: tjelesna masa ptica u pokusnim skupinama bila je značajno viša (P≤0,05), i to za 11 kg u drugoj skupini, za 13,3 kg u trećoj skupini i za 16,4 kg u četvrtoj skupini, u usporedbi s kontrolnom skupinom. Tijekom pokusa opažen je porast brojnosti eritrocita, leukocita te koncentracije hemoglobina, ali su ti, kao i ostali testirani hematološki pokazatelji, bili unutar fizioloških vrijednosti. Biokemijski krvni pokazatelji u svim skupinama nojeva nisu se značajno mijenjali na početku ni na kraju pokusa. No, razine ukupnih proteina, glukoze, Ca i P, pokazivale su rastući trend u svim pokusnim skupinama u korelaciji s dozom testiranog dodatka hrani. Dodatak hrani utjecao je na promjene u kemijskom sastavu i biološkoj vrijednosti mesa. U usporedbi s kontrolnom skupinom, zabilježen je porast u sadržaju pepela, masti, proteina i energije te u razini minerala, vitamina i nekih aminokiselina u mesu pokusnih nojeva, u postojanoj ovisnosti o udjelu dodatka hrani (P≤0,05). Zaključili smo da je uporaba dodatka hrani povoljno utjecala na rast, hematološke pokazatelje krvi, kao i na nutritivnu i biološku vrijednost mesa nojeva