Genomic analysis reveals the association of KIT and MITF variants with the white spotting in swamp buffaloes.

BACKGROUND: Swamp-type buffaloes with varying degrees of white spotting are found exclusively in Tana Toraja, South Sulawesi, Indonesia, where spotted buffalo bulls are highly valued in accordance with the Torajan customs. The white spotting depigmentation is caused by the absence of melanocytes. However, the genetic variants that cause this phenotype have not been fully characterized. The objective of this study was to identify the genomic regions and variants responsible for this unique coat-color pattern. RESULTS: Genome-wide association study (GWAS) and selection signature analysis identified MITF as a key gene based on the whole-genome sequencing data of 28 solid and 39 spotted buffaloes, while KIT was also found to be involved in the development of this phenotype by a candidate gene approach. Alternative candidate mutations included, in addition to the previously reported nonsense mutation c.649 C > T (p.Arg217*) and splice donor mutation c.1179 + 2T > A in MITF, a nonsense mutation c.2028T > A (p.Tyr676*) in KIT. All these three mutations were located in the genomic regions that were highly conserved exclusively in Indonesian swamp buffaloes and they accounted largely (95%) for the manifestation of white spotting. Last but not the least, ADAMTS20 and TWIST2 may also contribute to the diversification of this coat-color pattern. CONCLUSIONS: The alternative mutations identified in this study affect, at least partially and independently, the development of melanocytes. The presence and persistence of such mutations may be explained by significant financial and social value of spotted buffaloes used in historical Rambu Solo ceremony in Tana Toraja, Indonesia. Several de novo spontaneous mutations have therefore been favored by traditional breeding for the spotted buffaloes.

Impact of air pollution from different sources on sperm DNA methylation.

Environmental exposure is associated with increased incidence of respiratory and cardiovascular diseases and reduced fertility. Exposure to air pollution can influence gene expression through epigenetic mechanisms. In this study, we analysed gene-specific CpG methylation in spermatozoa of city policemen occupationally exposed to air pollution in two Czech cities differing by sources and composition of the air pollution. In Prague, the pollution is mainly formed by NO2 from heavy traffic. Ostrava is a hotspot of industrial air pollution with high concentrations of particular matter (PM) and benzo[a]pyrene (B[a]P). We performed genome-wide methylation sequencing using the SureSelectXT Human Methyl-Seq system (Agilent Technologies) and next-generation sequencing to reveal differentially methylated CpG sites and regions. We identified differential methylation in the region chr5:662169 - 663376 annotated to genes CEP72 and TPPP. The region was then analysed in sperm DNA from 117 policemen using targeted methylation sequencing, which proved its hypermethylation in sperm of Ostrava policemen.

Genome-wide analysis of homozygosity regions in european simmental bulls.

The study of Runs of Homozygosity (ROH) is a useful approach for the characterization of the genome of livestock populations. Due to their high relationship with autozygosity, ROH allow to make inference about population genetic history, to estimate the level of inbreeding, to assess within breed heterogeneity and to detect the footprints of selection on livestock genomes. Aim of this study was to investigate the distribution of runs of homozygosity in bulls belonging to five European Simmental populations and to assess the relationship between three production traits (milk yield, fat and protein contents) and autozygosity. ROH count, distribution and ROH-based coefficient of inbreeding (FROH ) were calculated for 3,845 Simmental bulls of five different European countries: Austria (AT), Switzerland (CH), Czech Republic (CZ), Germany (DE) and Italy (IT). Average values of ROH number per animal, and total genome length covered by ROH were 77.8 ± 20.7 and 205 ± 74.4 Mb, respectively. Bulls from AT, DE and IT exhibited similar ROH characteristics. Swiss animals showed the highest (12.6%), while CZ the lowest (4.6%) FROH coefficient. The relationship between ROH occurrence and milk production traits was investigated through a genome-wide ROH-traits association analysis (GWRA). A total of 34 regions previously associated with milk traits (yield and/or composition) were identified by GWRA. Results of the present research highlight a mixed genetic background in the 5 European Simmental populations, with the possible presence of three subgroups. Moreover, a strong relationship between autozygosity and production traits has been detected.

Cytogenetic Characterization of a Small Evolutionary Rearrangement Involving Chromosomes BTA21 and OAR18.

Both cattle (Bos taurus) and sheep (Ovis aries) belong to the Bovidae family but to different subfamilies, Bovinae and Caprinae, respectively. From a chromosomal point of view, apart from the already known centric fusions (that occurred during the evolutionary process in the Bovidae family) and the small differences in the chromosome classification, the 2 karyotypes are very similar in banding patterns. In this study, the combination of bioinformatics techniques and physical mapping of DNA markers enabled the identification of a micro-rearrangement, a small inversion involving bovine chromosome 21 (BTA21) and the corresponding sheep chromosome 18 (OAR18). The aim of this study was the cytogenetic characterization of this difference in genomic assemblies between cattle and sheep in this single chromosome region. To verify the inversion in FISH experiments, we used the BACs 442H08 and 222H03 from the INRA library and BACs 134H22 and 436P08 from the sheep-specific CHORI library. The results confirmed the presence of the inverted fragment in sheep compared to the cattle genome. Genomic rearrangements may have consequences depending on their influence on gene activity, but in this case no gene or transcribed DNA portion seemed to be involved. In conclusion, we showed for the first time, concerning autosomes, that besides the already known centric fusions also other differences exist between the bovine and sheep karyotypes. Furthermore, we demonstrated that the combination of a bioinformatics approach and physical mapping is a valid tool for the identification of currently unknown rearrangements between related species.

A de novo reciprocal chromosomal translocation t(3;6)(p14;q26) in the black Lucano pig.

In the past two decades, several cytogenetic screening programmes identified different chromosome rearrangements in pig, most of which represented by reciprocal translocation (rcp). This chromosome abnormality does not involve the variation in the number of chromosomes, but only the rearrangement of genetic material, resulting in phenotypically normal carriers with fertility problems. During an occasional cytogenetic screening, a new reciprocal translocation was detected in the black Lucano pig native breed. We analysed 15 animals reared by a family-run piggery in Basilicata region (Southern Italy). After karyotyping, four pigs (two boars and two sows) revealed two unpaired chromosomes. Analysis of the RBA karyotype and the dual-colour FISH technique confirmed that these pigs showed the same reciprocal translocation involving the chromosomes SSC3 and SSC6. The precise location of breakpoints was identified by RBH-FISH t(3;6)(p14;q26), whereas the analysis of the pedigree showed a case of Mendelian inheritance within a family, after the de novo occurrence of the new rcp. Considering the consequences of the rcp on the fertility, this study points out the importance of the cytogenetic screening in the native breeds for the safeguard of the genetic biodiversity and the sustainability of the rural areas.

Casein composition and differential translational efficiency of casein transcripts in donkey's milk.

The amount of the four caseins (αs1, αs2, ß and κ-CN) in donkey milk was evaluated by Urea-PAGE analysis at pH 8.6, followed by immuno-detection with polyclonal antibodies, coupled to densitometric analysis. The results showed the percentage of each casein in decreasing order: ß (54.28) > αs1 (35.59) > αs2 (7.19) > κ-CN (2.79). The mRNA quantification of donkey casein transcripts, carried out by RT-qPCR, showed that the average percentage of corresponding gene transcripts (CSN2, CSN1S1, CSN1S2 I and CSN3) was 70.85, 6.28, 14.23 and 8.65, respectively. The observed translation efficiency, assessed as percentage of single milk casein fraction out of single percentage of transcript, was 0.76, 5.66, 0.50 and 0.32, respectively. The analysis of the sequences flanking the start codon, the codon usage frequencies and the coding sequence length might explain, at least in part, the differential transcriptional and translational rate observed among the casein transcripts.

A fast and reliable polymerase chain reaction method based on short interspersed nuclear elements detection for the discrimination of buffalo, cattle, goat, and sheep species in dairy products.

OBJECTIVE: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the α-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). METHODS: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). RESULTS: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources (0.25 ng/µL). CONCLUSION: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

Role of X-linked inhibitor of apoptosis (XIAP) in frozen and thawed dormant and normal-hatched murine blastocysts.

Cryo-injury of mammalian blastocysts occurs during cryopreservation and induces apoptosis in trophoblast cells. This damage affects subsequent embryo development or may even cause death before implantation. X-linked inhibitor of apoptosis (XIAP) is an anti-apoptosis gene that has been widely studied in cancer research. However, only a few studies have investigated the activity of XIAP in cryopreservation. In this study, we investigate the role of XIAP in frozen and thawed murine blastocysts. A total of 1630 blastocysts were divided into fresh and freeze-thaw groups, and XIAP expression was investigated using qPCR, Western blot and confocal analyses. In addition, the effect of the embelin (a XIAP inhibitor) was also evaluated by co-culturing 390 dormant blastocysts. XIAP protein is primarily localized to the mitochondria of trophoblastic cells. Gene and protein expression is significantly down-regulated in blastocysts after cryopreservation, whereas embelin has negative effect on their survivals. These findings further broaden the understanding of mammalian embryonic cryopreservation.

Milk microRNA-146a as a potential biomarker in bovine tuberculosis.

In this research communication we exploited the potential use of milk microRNAs (miRs) as biomarkers for bovine tuberculosis (bTB). bTB is a zoonotic disease caused by Mycobacterium bovis which affects animal health, influencing herd economic sustainability. Diagnosis is based on skin delayed-type hypersensitivity reaction and quantification of interferon gamma but both techniques are influenced by several confounding factors. Thus, new methods for early diagnosis are required. In this context, microRNAs have been used as promising biomarkers for both infectious and non-infectious diseases. To determine the possible involvement of microRNAs in bTB, we analysed the expression of four immune-related miRs in 200 cows grouped in cases and controls with respect to positivity to tuberculosis. The analysis showed a different magnitude of expression in the groups indicating that active tuberculosis could influence miRs expression. We used expression values of miR-146a, the highest differentially expressed miR, for Receiver operating characteristic (ROC) curve analysis. In order to determine a test cut-off value for miR-146a expression that would differentiate cases and controls, a value for the miR-146a expression higher than 8 was selected as this gave a test specificity and sensitivity of 80·0% and 86·0% respectively. These values confirm the possibility of using miR-146a as a milk prognostic biomarker for bovine tuberculosis.

Molecular characterisation, genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5' flanking region and 478/477 nucleotides at the 3' flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

A novel polymorphism in the oxytocin receptor encoding gene (OXTR) affects milk fatty acid composition in Italian Mediterranean river buffalo.

The oxytocin receptor, also known as OXTR, is a protein which functions as receptor for the hormone and neurotransmitter oxytocin and the complex oxytocin-oxytocin receptor plays an important role in the uterus during calving. A characterisation of the river buffalo OXTR gene, amino acid sequences and phylogenetic analysis is presented. The DNA regions of the OXTR gene spanning exons 1, 2 and 3 of ten Mediterranean river buffalo DNA samples were analysed and 7 single nucleotide polymorphisms were found. We focused on the g.129C > T SNP detected in exon 3 and responsible for the amino acid replacement CGCArg > TGCCys in position 353. The relative frequency of T allele was of 0·257. An association study between this detected polymorphism and milk fatty acids composition in Italian Mediterranean river buffalo was carried out. The fatty acid composition traits, fatty acid classes and fat percentage of 306 individual milk samples were determined. Associations between OXTR g.129C > T genotype and milk fatty acids composition were tested using a mixed linear model. The OXTR CC genotype was found significantly associated with higher contents of odd branched-chain fatty acids (OBCFA) (P < 0·0006), polyunsaturated FA (PUFA n 3 and n 6) (P < 0·0032 and P < 0·0006, respectively), stearic acid (C18) (P < 0·02) and lower level of palmitic acid (C16) (P < 0·02). The results of this study suggest that the OXTR CC animals might be useful in selection toward the improvement of milk fatty acid composition.

Sister chromatid exchange test in river buffalo lymphocytes treated in vitro with furocoumarin extracts.

Furocoumarin extracts from Psoralea morisiana, the endemic Sardinian legume species, were tested for their mutagenic potential on river buffalo blood cells. The results obtained performing the sister chromatid exchange (SCE) test in blood cultures of five river buffalo calves (exposure to furocoumarins for 72h) and five cows (exposure to furocoumarins for 3h, in the absence and presence of S9 metabolic activator) are reported. Significant differences in mean values of SCEs were observed in cells of calves compared to control cells (unexposed), but no differences in SCE mean values were found between treated and untreated cells of cows in the presence or absence of S9. SCE mean values were much higher in cells of cows (exposed and control) than in cells of calves. Indeed, in calf cells, SCE mean values/cell (±SD) were 6.66±2.45 in the control and 7.63±3.01, 9.03±3.90, 9.53±3.60 and 9.99±3.41 in treated cells at 50, 100, 200 and 400 µg/ml of furocoumarin extracts, respectively. In cow cells, grown in presence of S9, SCE mean values/cell were 11.49±4.78 and 11.65±5.19 in treated cells at 100 and 200 µg/ml of furocoumarins and 11.66±5.45 in the control. In cow cells grown in absence of S9, SCE mean values were 11.81±6.14 in the control and 12.35±7.09 and 12.01±5.43, respectively, in the presence of 100 and 200 µg/ml of furocoumarins. Despite their higher SCE values in the absence of S9, no statistically significant differences were found when these values were compared with those shown in presence of S9, suggesting no mutagenic action of furocoumarins in cows, at the doses used in this study.

Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

Fatal Outcome in a Newborn Calf Associated with Partial Trisomy 25q and Partial Monosomy 11q, 60,XX,der(11)t(11;25)(q11;q14∼21).

A newborn calf of the Agerolese cattle breed underwent clinical cytogenetic investigation because of hyperflexion of the forelimbs, red eyes and the inability to stand. Anamnesis revealed that the mother, phenotypically normal, carried a chromosomal aberration. The newborn died after 2 weeks, and no remarkable alterations were found by the veterinarian on postmortem examination. The mother was a carrier of a reciprocal balanced translocation rcp(11;25)(q11,q14∼21) detected after a cytogenetic investigation in 2011; however, the analysis of the newborn revealed a different chromosomal aberration with partial trisomy of chromosome 25 and partial monosomy of chromosome 11. In fact, the results showed both chromosomes 25, one chromosome 11 and only one long derivative chromosome (der11). FISH analysis, performed using BAC clones, confirmed the chromosomes and their regions involved. Finally, both the localization of the breakpoints on band q11 (centromere) of chromosome 11 and band q14-21 of chromosome 25, and the complete loss of the der25 identified the aberration as an unbalanced translocation 60,XX,der(11)t(11;25)(q11;q14∼21). A comparison with human chromosomes was also performed to search for similarities and possible genes involved in order to study their effects, thus extending the knowledge of these aberrations by case reports.

The utility of chromosome microdissection in clinical cytogenetics: a new reciprocal translocation in sheep.

Local sheep breeders and scientists in Italy cooperate and conduct research on the genetic improvement of autochthonous genetic types (AGTs) by various approaches, including a cytogenetic breeding selection since 2011. The Laticauda sheep (Ovis aries, 2n = 54) breed is one of the AGTs reared in the Campania region (southern Italy). Performing cytogenetic analyses, we have detected and described a novel reciprocal translocation in a Laticauda sheep identified as 54,XX t(18;23)(q14;q26). Our data support recurring appeals that suggest the regular performance of cytogenetic analyses for monitoring genetic health of livestock species. In total, 5 cases of reciprocal translocations in sheep are known, including the new case. None of them has any phenotypic effect on the living offspring. However, affected animals are characterized by sterility or have a low fertility which can have an effect on breeding success and on economical balance. Presence and kind of the described novel chromosomal aberration were detected by performing CBA-banding and FISH mapping with telomeric probes. RBA-banding allowed the karyotyping of sheep chromosomes and the identification of aberrant chromosomes and regions involved in the new reciprocal translocation. Whole chromosome painting (WCP) probes received from equivalent chromosomes in cattle and the derivative sheep chromosome 18 confirmed the cytogenetic data. This way, our study underlined both the importance of WCP probes by chromosome microdissection and a new way to use WCP probes directly generated from derivative chromosomes.

CSN1S1, CSN3 and LPL: Three Validated Gene Polymorphisms Useful for More Sustainable Dairy Production in the Mediterranean River Buffalo.

The search for DNA polymorphisms useful for the genetic improvement of dairy farm animals has spanned more than 40 years, yielding relevant findings in cattle for milk traits, where the best combination of alleles for dairy processing has been found in casein genes and in DGAT1. Nowadays, similar results have not yet been reached in river buffaloes, despite the availability of advanced genomic technologies and accurate phenotype records. The aim of the present study was to investigate and validate the effect of four single nucleotide polymorphisms (SNP) in the CSN1S1, CSN3, SCD and LPL genes on seven milk traits in a larger buffalo population. These SNPs have previously been reported to be associated with, or affect, dairy traits in smaller populations often belonging to one farm. A total of 800 buffaloes were genotyped. The following traits were individually recorded, monthly, throughout each whole lactation period from 2010 to 2021: daily milk yield (dMY, kg), protein yield (dPY, kg) and fat yield (dFY, kg), fat and protein contents (dFP, % and dPP, %), somatic cell count (SCC, 103 cell/mL) and urea (mg/dL). A total of 15,742 individual milk test day records (2496 lactations) were available for 680 buffalo cows, with 3.6 ± 1.7 parities (from 1 to 13) and an average of 6.1 ± 1.2 test day records per lactation. Three out four SNPs in the CSN1S1, CSN3 and LPL genes were associated with at least one of analyzed traits. In particular, the CSN1S1 (AJ005430:c.578C>T) gave favorable associations with all yield traits (dMY, p = 0.022; dPY, p = 0.014; dFY, p = 0.029) and somatic cell score (SCS, p = 0.032). The CSN3 (HQ677596: c.536C>T) was positively associated with SCS (p = 0.005) and milk urea (p = 0.04). Favorable effects on daily milk yield (dMY, p = 0.028), fat (dFP, p = 0.027) and protein (dPP, p = 0.050) percentages were observed for the LPL. Conversely, the SCD did not show any association with milk traits. This is the first example of a confirmation study carried out in the Mediterranean river buffalo for genes of economic interest in the dairy field, and it represents a very important indication for the preselection of young bulls destined for breeding programs aimed at more sustainable dairy production.

Factors Affecting the Milk Production Traits and Lactation Curve of the Indigenous River Buffalo Populations in Bangladesh.

Household buffalo dairy farming is gaining popularity nowadays in Bangladesh because of the outstanding food value of buffalo milk as well as the lower production cost of buffalo compared to cattle. An initiative has recently been taken for the genetic improvement of indigenous dairy buffaloes. The present study was carried out to determine the influence of some environmental factors like age, parity, season of calving, calving interval, dry period on the lactation yield, and lactation curve of indigenous dairy buffaloes of Bangladesh. A total of 384 indigenous dairy buffaloes from the 3rd and 4th parity of seven herds under two different agroecological zones covering four seasons were selected and ear tagged for individual buffalo milk recording. A milk yield of 300 days (MY300d) was calculated following the International Committee for Animal Recording (ICAR) and the data were evaluated using the generalized linear model (GLM). In production traits, the mean of calculated lactation period (CLP), calculated lactation yield (CLY), and milk yield of 300 days (MY300d) of the overall population were 267.28 days, 749.36 kg, and 766.92 kg, respectively, whereas calving interval (CI) and dry period (DP) as reproductive traits were 453.06 days and 185.78 days, respectively. The season of calving, age of buffalo cows, population or herd, agroecological zone, calving interval, and dry period had significant effects on production traits (p < 0.05 to p < 0.001). The season of calving, level of milk production of 300 days, population, and agroecological zone significantly affected the reproduction traits (p < 0.01 to p < 0.001). Parity was found to be non-significant for both types of traits. The average peak yield of test day (TD) milk production was highest at TD4 (4.47 kg, 98th day of lactation). The average MY300d of milk production was the highest in the Lalpur buffalo population (1076.13 kg) and the lowest in the buffalo population of Bhola (592.44 kg). The correlations between milk production traits (CLP, CLY, and MY-300d) and reproduction traits (CI and DP) were highly significant (p < 0.01 to p < 0.001). Positive and high correlation was found within milk traits and reproduction traits, but correlation was negative between milk traits and reproduction traits. Therefore, these non-genetic factors should be considered in the future for any genetic improvement program for indigenous dairy buffaloes in Bangladesh.

A Circular RNA Generated from Nebulin (NEB) Gene Splicing Promotes Skeletal Muscle Myogenesis in Cattle as Detected by a Multi-Omics Approach.

Cattle and the draught force provided by its skeletal muscle have been integral to agro-ecosystems of agricultural civilization for millennia. However, relatively little is known about the cattle muscle functional genomics (including protein coding genes, non-coding RNA, etc.). Circular RNAs (circRNAs), as a new class of non-coding RNAs, can be effectively translated into detectable peptides, which enlightened us on the importance of circRNAs in cattle muscle physiology function. Here, RNA-seq, Ribosome profiling (Ribo-seq), and peptidome data are integrated from cattle skeletal muscle, and detected five encoded peptides from circRNAs. It is further identified and functionally characterize a 907-amino acids muscle-specific peptide that is named circNEB-peptide because derived by the splicing of Nebulin (NEB) gene. This peptide localizes to the nucleus and cytoplasm and directly interacts with SKP1 and TPM1, key factors regulating physiological activities of myoblasts, via ubiquitination and myoblast fusion, respectively. The circNEB-peptide is found to promote myoblasts proliferation and differentiation in vitro, and induce muscle regeneration in vivo. These findings suggest circNEB-peptide is an important regulator of skeletal muscle regeneration and underscore the possibility that more encoding polypeptides derived by RNAs currently annotated as non-coding exist.