Jolanta Oprządek

Transcriptional background of beef marbling — Novel genes implicated in intramuscular fat deposition

The purpose of this study was to identify novel marbling-related genes by comparison of the global gene expression in semitendinosus muscle of 15-month-old Limousin (LIM), Holstein-Friesian (HF) and Hereford (HER) bulls. Muscle of LIM was lean with low intramuscular fat (IMF) content (0.53%) unlike the marbled muscles of HER and HF characterized by higher amounts of IMF (1.10 and 0.81%, respectively). The comparison of muscle transcriptional profile between marbled and lean beef revealed significant differences in expression of 144 genes, presumably involved in consecutive stages of adipose tissue development, such as preadipocyte proliferation and differentiation, adipocyte maturation, lipid filling and lipid metabolism leading to increased IMF deposition and marbling development. Correlation coefficients and regression analysis for nine of them (gadd45a, pias3, ccrn4l, diras3, pou5f1, hoxa9, atp2a2 and pim1) validated by real-time qPCR confirmed their moderate-high correlation with IMF% and explained up to 70.5% of the total variability in IMF deposition in the bulls.

Comparison of skeletal muscle transcriptional profiles in dairy and beef breeds bulls

A cDNA microarray (18 263 probes) was used for transcriptome analysis of bovine skeletal muscle (m. semitendinosus) in 12-month-old bulls of the beef breed Limousin (LIM) and the typical dairy breed Holstein-Friesian (HF, used as a reference). We aimed to identify the genes whose expression may reflect the muscle phenotype of beef bulls. A comparison of muscle transcriptional profiles revealed significant differences in expression of 393 genes between HF and LIM. We classified biological functions of 117 genes with over 2-fold differences in expression between the examined breeds. Among them, 72 genes were up-regulated and 45 genes were down-regulated in LIM vs. HF. The genes were involved in protein metabolism and modifications (22 genes), signal transduction (15), nucleoside, nucleotide and nucleic acid metabolism (13), cell cycle (9), cell structure and motility (9), developmental processes (9), intracellular protein traffic (7), cell proliferation and differentiation (6), cell adhesion (6), lipid, fatty acid and steroid metabolism (5), transport (5), and other processes. For the purpose of microarray data validation, we randomly selected 4 genes:trip12, mrps30, pycrl, andc-erbb3. Real-time RT-PCR results showed similar trends in gene expression changes as those observed in microarray studies. Basing on results of the present study, we proposed a model of the regulation of skeletal muscle growth and differentiation, with a principal role of the somatotropic pathway. It may explain at least in part the development of muscle phenotype in LIM bulls. We assume that the growth hormone directly or indirectly (through IGF-1) activates the calcium-signaling pathway with calcineurin, which stimulates myogenic regulatory factors (MRFs) and inhibits early growth response gene. The inhibition results in indirect activation of MRFs and impaired activation of TGF-beta1 and myostatin, which finally facilitates terminal muscle differentiation.

Gene expression profiling in skeletal muscle of Holstein-Friesian bulls with single-nucleotide polymorphism in the myostatin gene 5'-flanking region

Myostatin (GDF-8) is a key protein responsible for skeletal muscle growth and development, thus mutations in themstn gene can have major economic and breeding consequences. The aim of the present study was to investigate myostatin gene expression and transcriptional profile in skeletal muscle of Holstein-Friesian (Black-and-White) bulls carrying a polymorphism in the 5’-flanking region of themstn gene (G/C transversion at position -7828). Real-time qRT-PCR and cDNA microarray revealed significantly lowermstn expression in muscle of bulls with the CC genotype, as compared to GG and GC genotypes. The direct comparison of skeletal muscle transcriptional profiles between the CC genotype and GG and GC genotypes resulted in identification of genes, of which at least some can be putative targets for myostatin. Using cDNA microarray, we identified 43 common genes (includingmstn) with significantly different expression in skeletal muscle of bulls with the CC genotype, as compared to GG and GC genotypes, 15 of which were upregulated and 28 were downregulated in the CC genotype. Classification of molecular function of differentially expressed genes revealed the highest number of genes involved in the expression of cytoskeleton proteins (9), extracellular matrix proteins (4), nucleic acid-binding proteins (4), calcium-binding proteins (4), and transcription factors (4). The biological functions of the largest number of genes involved: protein metabolism and modification (10), signal transduction (10), cell structure (8), and developmental processes (8). The main identified signaling pathways were: Wnt (4), chemokines and cytokines (4), integrin (4), nicotine receptor for acetylocholine (3), TGF-beta (2), and cytoskeleton regulation by Rho GTPase (2). We identified previously unrecognized putatively myostatin-dependent genes, encoding transcription factors (EGR1, Nf1b, ILF1), components of the proteasomal complex (PSMB7, PSMD13) and proteins with some other molecular function in skeletal muscle (ITGB1BP3, Pla2g1b, ISYNA1, TNFAIP6, MST1, TNNT1, CALB3, CACYBP, and CTNNA1).

Genetic basis of mastitis resistance in dairy cattle - a review* *

Abstract Mastitis is one of the most important mammary gland diseases impacting lactating animals. Resistance to this disease could be improved by breeding. There are several selection methods for mastitis resistance. To improve the natural genetic resistance of cows in succeeding generations, current breeding programmes use somatic cell count and clinical mastitis cases as resistance traits. However, these methods of selection have met with limited success. This is partly due to the complex nature of the disease. The limited progress in improving udder health by conventional selection procedures requires applying information on molecular markers of mastitis susceptibility in marker-assisted selection schemes. Mastitis is under polygenic control, so there are many genes that control this trait in many loci. This review briefly describes genome-wide association studies which have been carried out to identify quantitative trait loci associated with mastitis resistance in dairy cattle worldwide. It also characterizes the candidate gene approach focus on identifying genes that are strong candidates for the mastitis resistance trait. In the conclusion of the paper we focus our attention on future research which should be conducted in the field of the resistance to mastitis. Streszczenie Zapalenie wymienia (mastitis) jest jedną z najważniejszych chorób bydła mlecznego. Poprawa odporności na tę chorobę jest możliwa dzięki wykorzystaniu metod hodowlanych. Aby poprawić odporność krów mlecznych na mastitis, dopuszczalne jest wykorzystanie kilku metod selekcyjnych, które zostały omówione pokrótce w niniejszej pracy. Programy hodowlane, obecnie stosowane na świecie, wykorzystują jako kryterium selekcyjne zwierząt odpornych na mastitis, liczbę komórek somatycznych w mleku krów lub częstość występowania przypadków klinicznych. Programy te jednak tylko częściowo przyczyniły się do poprawy odporności krów na zapalenie wymienia. Jednym z powodów małej ich efektywności jest złożona etiologia tej choroby. Aby zwiększyć efektywność programów hodowlanych w odniesieniu do odporności na mastitis, w ostatnich latach podjęto badania nad markerami genetycznymi tej choroby i ich wykorzystaniem w selekcji wspomaganej markerami. Na zapalenie wymienia wpływa wiele genów. W niniejszej pracy dokonano przeglądu badań nad poszukiwaniem markerów związanych z odpornością na mastitis i genów do niej kandydujących. W podsumowaniu wskazano kierunki dalszych badań.

Expression in promoter variant of the ERα gene in bos taurus liver

Due to the variant functions that estrogens play in the regulation of reproduction, development of the mammary gland, growth and differentiation of cells, estrogen receptors and their genes are considered as a candidates for the markers of production and functional traits in farm animals, including cattle. In the earliest study, a 2853-bp bovine ER gene 5′-region was PCR amplified and sequenced. Moreover, for the first time, a polymorphism was described within 5′ region of the bovine ERα gene—A/G transition lying upstream at position 2591 from acceptor splice site +85, possibly within its promoter—which could be recognized with RFLP-BglI. In other study we are found second polymorphism—A/G transition at position 1213 from acceptor splice site +85, located in promoter for exon B. We have examined the specific mRNA expression of ERα in various genotypes using real-time RT-PCR. We used four animals from each genotype group—AG, GG for BglI and AA, AG for SnaBI—to analyse liver ERα expression at the level of Real-time PCR. Liver samples were taken from the 16 young Friesian bulls of the different ERα genotypes, slaughtered at the local abattoir. As shown by Real-Time PCR, on the livers of animals with different genotype ERα mRNA for BglI polymorphism we didn’t found variability, but for SnaBI we have found variability between AG and AA genotypes.

Genetic disorders in beef cattle: a review

The main purpose of present review is to describe and organize autosomal recessive disorders (arachnomelia, syndactylism, osteopetrosis, dwarfism, crooked tail syndrome, muscular hyperplasia, glycogen storage disease, protoporphyria), which occur among beef cattle, and methods that can be applied to detect these defects. Prevalence of adverse alleles in beef breeds happens due to human activity—selections of favorable features, e.g. developed muscle tissue. Unfortunately, carriers of autosomal recessive diseases are often characterized by these attributes. Fast and effective identification of individuals, that may carry faulty genes, can prevent economical losses.

Breed-dependent microRNA expression in the primary culture of skeletal muscle cells subjected to myogenic differentiation

BackgroundSkeletal muscle in livestock develops into meat, an important source of protein and other nutrients for human consumption. The muscle is largely composed of a fixed number of multinucleated myofibers determined during late gestation and remains constant postnatally. A population of postnatal muscle stem cells, called satellite cells, gives rise to myoblast cells that can fuse with the existing myofibers, thus increasing their size. This requires a delicate balance of transcription and growth factors and specific microRNA (miRNA) expressed by satellite cells and their supporting cells from the muscle stem cell niche. The role of transcription and growth factors in bovine myogenesis is well-characterized; however, very little is known about the miRNA activity during this process. We have hypothesized that the expression of miRNA can vary between primary cultures of skeletal muscle cells isolated from the semitendinosus muscles of different cattle breeds and subjected to myogenic differentiation.ResultsAfter a 6-day myogenic differentiation of cells isolated from the muscles of the examined cattle breeds, we found statistically significant differences in the number of myotubes between Hereford (HER)/Limousine (LIM) beef breeds and the Holstein-Friesian (HF) dairy breed (p ≤ 0.001). The microarray analysis revealed differences in the expression of 23 miRNA among the aforementioned primary cultures. On the basis of a functional analysis, we assigned 9 miRNA as molecules responsible for differentiation progression (miR-1, -128a, -133a, -133b, -139, -206, -222, -486, and -503). The target gene prediction and functional analysis revealed 59 miRNA-related genes belonging to the muscle organ development process.ConclusionThe number of myotubes and the miRNA expression in the primary cultures of skeletal muscle cells derived from the semitendinosus muscles of the HER/LIM beef cattle breeds and the HF dairy breed vary when cells are subjected to myogenic differentiation. The net effect of the identified miRNA and their target gene action should be considered the result of the breed-dependent activity of satellite cells and muscle stem cell niche cells and their mutual interactions, which putatively can be engaged in the formation of a larger number of myotubes in beef cattle-related cells (HER/LIM) during in vitro myogenesis.

Lactoferrin gene variants, their expression in the udder and mastitis susceptibility in dairy cattle

Lactoferrin gene (LF) is regarded as one of the potential markers of mastitis susceptibility/resistance in dairy cattle. The study’s aim was therefore, to investigate the feasibility of two single nucleotide polymorphisms (SNP), placed in the 5′-flanking region and 3′-untranslated region of the LF gene, to serve as mastitis markers. The associations between these SNP and the expression of LF, both on mRNA and protein level, were estimated in the milk of Polish Holstein-Friesian cows. The relationships between polymorphisms and cows’ estimated breeding values (EBV) for somatic cell count were also calculated. It was shown that both polymorphisms have a significant impact on lactoferrin content in milk, and that LF+32 SNP is associated with the cow’s EBV for somatic cell count. No association between SNP chosen for the study and lactoferrin mRNA abundance in milk somatic cells was observed. We propose LF+32 SNP for a molecular marker of mastitis resistance in dairy cows.