PLASMA-ENHANCED CHEMICAL VAPOUR DEPOSITION ON PARTICLES IN AN ATMOSPHERIC CIRCULATING FLUIDIZED BED

Plasma-enhanced chemical vapour deposition is an attractive technqiue to provide particles with a thin film. Applying a cold plasma enables us to work with temperature-sensitive materials. Using a CFB with an incorporated volume dielectric barrier discharge reactor we coated 20-30 m CuO particles with a thin SiOx layer

SIRT1 gene methylation in sperm differs in rams with high and low fertility

Submitted 2020-07-02 | Accepted 2020-09-04 | Available 2020-12-01https://doi.org/10.15414/afz.2020.23.mi-fpap.156-161Recently, more evidences of epigenetic impact on the male fertility, particularly on sperm DNA methylation have been reported. Data related to this issue in livestock males is still limited. The present study analyzed the DNA methylation status of the important gene for spermatogenesis, SIRT1, in ram sperm and its correspondence with semen quality and fertilizing ability. The ejaculates of 10 rams (5 rams - 1.5 years old, and 5 rams - 4 years old) from Synthetic Population Bulgarian Milk breed were evaluated and used for the artificial insemination of 174 ewes in breeding season. Two semen samples from each animal were used for DNA extraction followed by bisulfite conversion. The DNA methylation status of SIRT1 was detected through quantitative methylation-specific PCR using two sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. On the base of age and conception rate the rams were divided in different groups. Data of semen quality, DNA methylation status of SIRT1 and reproductive performances of each group were statistically processed. Results showed a high average value of DNA methylation of SIRT1 in ram sperm (78.5±23.9%) and wide individual variability among investigated animals, with a coefficient of variation of 34.4%. The 1.5 years old animals tended to have a higher level of SIRT1 methylation than 4 years old animals. The rams in group with high fertilizing ability had significantly higher DNA methylation of SIRT1 in sperm than those with low fertilizing ability. In conclusion, results of this study provided evidence that the alteration of sperm SIRT1 methylation is associated with fertility performances of the rams and, probably, with their age.Keywords: sperm DNA methylation, SIRT1, ram fertilityReferencesAHLAWAT, S. et al. (2019). Promoter methylation and expression analysis of Bvh gene in bulls with varying semen motility parameters. Theriogenology, 125, 152–156. https://doi.org/10.1016/j.theriogenology.2018.11.001ASTON, K. I. et al. (2015). Aberrant sperm DNA methylation predicts male fertility status and embryo quality. Fertility and Sterility, 104, 1388–1397. https://doi.org/10.1016/j.fertnstert.2015.08.019AX, R. L. et al. (2000). Semen evaluation. In: Hafez, B., Hafez, E. S. E. (Eds.), Reproduction in Farm Animals, 7 th ed. Lippincott Williams and Wilkins, Philadelphia, pp. 365-375.BELL, E. L. et al. (2014). SirT1 is required in the male germ cell for differentiation and fecundity in mice. Development, 141(18), 3495-3504. https://doi.org/10.1242/dev.110627BOISSONNAS, C. C. et al. (2013). Epigenetic disorders and male subfertility. Fertility and Sterility, 99, 624–631. https://doi.org/10.1016/j.fertnstert.2013.01.124CONGRAS, A. et al. (2014). Sperm DNA methylation analysis in swine reveals conserved and species-specific methylation patterns and highlights an altered methylation at theGNAS locus in infertile boars. Biology of Reproduction, 91(6), 137, 1–14. https://doi.org/10.1095/biolreprod.114.119610COUSSENS, M. et al. (2008). Sirt1 Deficiency Attenuates Spermatogenesis and Germ Cell Function. PLoS ONE, 3(2), e1571. https://doi.org/10.1371/journal.pone.0001571DONKIN, I. and BARRES, R. (2018). Sperm epigenetics and influence of environmental factors. Molecular Metabolism, 14, 1–11. https://doi.org/10.1016/j.molmet.2018.02.006ISLAM, S. et al. (2020). DNA hypermethylation of sirtuin 1 (SIRT1) caused by betel quid chewing—a possible predictive biomarker for malignant transformation. Clinical Epigenetics, 12, 12. https://doi.org/10.1186/s13148-019-0806-y JENKINS, T. G. et al. (2019). Age-associated sperm DNA methylation patterns do not directly persist trans-generationally. Epigenetics & Chromatin 12,(1), NA https://doi.org/10.1186/s13072-019-0323-4JING, H. and LIN, H. (2015). Sirtuins in epigenetic regulation. Chemical Reviews, 115, 2350−2375. http://dx.doi.org/10.1021/cr500457hKENNEDY, D. (2012). Sheep Reproduction Basics and Conception Rates. http://www.omafra.gov.on.ca/english/livestock/sheep/facts/12-037.htmKROPP, J. et al. (2017). Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos. BMC Genomics, 18, 280. https://doi.org/10.1186/s12864-017-3673-yLAMBERT, S. et al. (2018). Spermatozoa DNA methylation patterns differ due to peripubertal age in bulls. Theriogenology, 106, 21–29. https://doi.org/10.1016/j.theriogenology.2017.10.006LAQQAN, M. et al. (2017). Alterations in sperm DNA methylation patterns of oligospermic males. Reproductive Biology, 17, 396–400. https://doi.org/10.1016/j.repbio.2017.10.007LIU, C. et al. (2017). Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice. Development, 144, 441-451. https://doi.org/10.1242/dev.147074MARTTILA, S. (2016). Ageing-associated Changes in Gene Expression and DNA Methylation. Academic dissertation. University of Tampere. https://trepo.tuni.fi/bitstream/handle/10024/98917/978-952-03-0073-9.pdfMCSWIGGIN, H. M. and O’DOHERTY, A. M. (2018). Epigenetic reprogramming during spermatogenesis and male factor infertility. Reproduction, 156, R9–R21. https://doi.org/10.1530/rep-18-000MOLARO, A. et al. (2011). Sperm methylation profiles reveal features of epigenetic inheritance and evolution in primates. Cell, 146, 1029–1041. https://doi.org/10.1016/j.cell.2011.08.016NAYAK, K. et al. (2016). Epigenetic regulation of gene expression during spermatogenesis. https://digitalcommons.uri.edu/srhonorsprog/491/.OAKES, C. C. et al. (2007). Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells. Developmental Biology, 307, 368–379. https://doi.org/10.1016/j.ydbio.2007.05.002OLIVIER, W. J. (2014). Calculation of reproduction parameters. Info pack ref: AP 2014/032, Grootfontein Agricultural Development Institute.PERRIER, J. P. et al. (2018). A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features. BMC Genomics, 19, 404. https://doi.org/10.1186/s12864-018-4764-0RAHMAN S. and ISLAM, R. (2011). Mammalian Sirt1: insights on its biological functions. Cell Communication and Signaling, 9, 11. https://doi.org/10.1186/1478-811X-9-11SHARAFI, M. et al. (2017). Epigenetic modulation of ram sperm during cryopreservation. Reproduction in Domestic Animals 52(S3), 133. https://doi.org/10.1111/rda.13026SCHAGDARSURENGIN, U. and STEGER, K. (2016). Epigenetics in male reproduction: effect of paternal diet on sperm quality and offspring health. Nature Reviews Urology, 13, 584–595. https://doi.org/10.1038/nrurol.2016.157SHOJAEI SAADI, H. A. et al. (2017). Genome-wide analysis of sperm DNA methylation from monozygotic twin bulls. Reproduction, Fertility and Development, 29, 838–843. https://doi.org/10.1071/rd15384TAKEDA, K. et al. 2019. Age-related changes in DNA methylation levels at CpG sites in bull spermatozoa and in vitro fertilization-derived blastocyst-stage embryos revealed by combined bisulfite restriction analysis. Journal of Reproduction and Development, 65, 305–312. https://doi.org/10.1262/jrd.2018-146TANG, Q. et al. (2017). Idiopathic male infertility and polymorphisms in the DNA methyltransferase genes involved in epigenetic marking. Scientific Reports, 7, 11219. https://doi.org/10.1038/s41598-017-11636-98TIBARY, A. et al. (2018). Ram and buck breeding soundness examination. Revue Marocaine des Sciences Agronomiques et Vétérinaires, 6(2), 241-255.TOLIC, A. et al. (2019). Absence of PARP‐1 affects Cxcl12 expression by increasing DNA demethylation. Journal of Cellular and Molecular Medicine, 23, 2610–2618. https://dx.doi.org/10.1111/jcmm.14154URDINGUIO, R. G. et al. (2015). Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility. Human Reproduction, 30,(5), 1014–1028. https://doi.org/10.1093/humrep/dev053VERMA, A. et al. (2014). Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility. Theriogenology, 82, 750–759. https://doi.org/10.1016/j.theriogenology.2014.06.012WOLFFE, A. P. and GUSCHIN, D. (2000). Review: chromatin structural features and targets that regulate transcription. Journal of Structural Biology, 129, 102–122. https://doi.org/10.1006/jsbi.2000.4217ZHOU, Y. et al. (2018). Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm. GigaScience, 7(5), giy039. https://doi.org/10.1093/gigascience/giy039

ПРОУЧВАНЕ ВЛИЯНИЕТО НА СУХ ЕКСТРАКТ ОТ TRIBULUS TERRESTRIS ВЪРХУ ОСНОВНИ БИОХИМИЧНИ И ХЕМАТОЛОГИЧНИ ПОКАЗАТЕЛИ НА КРЪВТА ПРИ ТОКАЧКИ (NUMIDA MELEAGRIS)

The aim of the present research was to investigate the effect of Bulgarian additive Vemoherb-T (dry extract of the annual plant Tribulus terrestris – L), produced by Vemo 99 Ltd Company, Sofia on main biochemical characteristics and hematological parameters of the blood in guinea fowl (Numida meleagris). An experiment was carried out with 30 Pearl-gray Guinea fowl (32 weeks old), distributed in two groups – a control and an experimental, 12 female and 3 male each. All birds were fed the same mixture for breeder guinea fowl. Vemoherb-T was supplemented to the compound feed of the experimental group in a daily dose of 10 mg/kg body weight for a period of 12 weeks. The tested product decreased significantly the levels of total triglycerides (P < 0.05), total cholesterol (P <0.01) and glucose (P < 0.01; P < 0.001 in male and female birds respectively) in the blood serum. It was established significantly higher total protein- ((P < 0.001) and calcium (P<0.01) values in the blood serum of the treated birds. The addition of Vemoherb-T increased significantly hemoglobin level, the number of erythrocytes and leukocytes and decreased the number of eosinophils in guinea fowl from the both sexes.Целта на настоящото изследване беше да се установи влиянието на Българския продукт Vemoherb-T (сух екстракт от едногодишното растение Tribulus terrestris-L), произведен от фирма Вемо 99 ООД, София, върху основни биохимични характеристики и хематологични показатели на кръвен серум при токачки (Numida meleagris). Беше проведен научен експеримент с 30 Бисерносиви токачки (на 32 седмична възраст), разпределени в две групи – опитна и контролна, 12 женски и 3 мъжки всяка. Всички птици получаваха една и съща смеска за разплодни токачки. Vemoherb-T беше добавян към комбинирания фураж на опитната група ежедневно в доза 10 mg/kg жива маса в продължение на 12 седмици. Изпитваният продукт понижава достоверно нивата на общите триглицериди (P < 0.05), общия холестерол (P <0.01) и глюкозата (P < 0.01; P < 0.001 съответно за мъжките и женски птици) в кръвния серум. Бяха установени достоверно по- високи стойности на общия протеин ((P < 0.001) и на калция (P < 0.01) в кръвния серум на третираните птици. Добавката на Vemoherb-T повишава достоверно нивото на хемоглобина, броя на еритроцитите и лeвкоцитите, и понижава броя на еозинофилните левкоцити при токачките и от двата пола

Oregonin from Alnus incana bark affects DNA methyltransferases expression and mitochondrial DNA copies in mouse embryonic fibroblasts

Oregonin is an open-chain diarylheptanoid isolated from Alnus incana bark that possesses remarkable antioxidant and anti-inflammatory properties, inhibits adipogenesis, and can be used in the prevention of obesity and related metabolic disorders. Here, we aimed to investigate the effects of oregonin on the epigenetic regulation in cells as well as its ability to modulate DNA methylating enzymes expression and mitochondrial DNA (mtDNA) copies. Our results show that oregonin altered the expression of DNA methyltransferases and mtDNA copy numbers in dependency on concentration and specificity of cells genotype. A close correlation between mtDNA copy numbers and mRNA expression of the mtDnmt1 and Dnmt3b was established. Moreover, molecular modeling suggested that oregonin fits the catalytic site of DNMT1 and partially overlaps with binding of the cofactor. These findings further extend the knowledge on oregonin, and elucidate for the first time its potential to affect the key players of the DNA methylation process, namely DNMTs transcripts and mtDNA

DNA Methylation Level of Gene SIRT1 in Ram Spermatozoa and Relationship with Fertilizing Ability According to Breed and Age

Background: Effect of the epigenetic factors on the male fertility is well proofed. Sperm acts as a carrier of genetic material, and its DNA methylome can affect maternal pregnancy rate and offspring phenotype. However, the research on the DNA methylation in the spermatozoids of livestock males, in particular rams, is still limited. To best of our knowledge the data about as a global as well as gene specific DNA methylation in ram spermatozoa from different breeds and ages are missed in the scientific literature. The present study was designed to analyze the relationship between methylation levels of the important for spermatogenesis gene SIRT1 in spermatozoa and fertilizing ability of sperm in rams from different breeds and ages. Materials, Methods &amp; Results: The ejaculates of 16 rams from Lacaune, East Friesian and Assaf breeds at age between 18 to 96 months were evaluated. The kinematic parameters of 2 semen samples from each animal were estimated by CASA. The separated spermatozoa were used for DNA extraction followed by bisulfite conversion. The DNA methylation of SIRT1 was detected through quantitative methylation-specific PCR using 2 sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. The breed and age effect on the gene SIRT1 methylation by ANOVA was estimated. Experimental females included 393 clinically healthy milk ewes (Lacaune, n = 131; East Friesian sheep, n = 100 and Assaf, n = 162) in breeding season. Reproductive performances (conception rate at lambing, lambing percentage and fecundity) of ewes, inseminated by sperm of the investigated rams, were statistically processed. ANOVA showed that the animal breed influences significantly on the level of DNA methylation of gene SIRT1 in ram spermatozoa (P = 0.002) An average value of DNA methylation of SIRT1 in ram sperm from Lacaune breed was significantly higher than in Assaf and East Friesian (81.21 ± 15.1% vs 36.7 ± 14.2% and 38.3 ± 18.6 respectively, P &lt; 0.01). The highest percent of SIRT1 methylation was observed in old animals compared to the young and middle-age. Moderate and strong correlations (r from 0.44 to 0.71, P &lt; 0.05) between the methylation level of the SIRT1 gene in rams' sperm and reproductive parameters of inseminated ewes in all breeds were established. Discussion: Our data are the first message about the effect of breed on the specificity of DNA methylation of gene SIRT1 in ram spermatozoa. These results demonstrated an existence of the sheep breeds with high and low level of DNA methylation of gene SIRT1 in ram sperm. Although the effect of age on the methylation level in sperm is still discussable, our results showed a moderate correlation between age and methylation level of SIRT1 in spermatozoa of rams. Taking into account that DNA methylation in sperm is stabilized with puberty onset and is a heritable epigenetic modification, it can be a promising marker of sperm quality in animal breeding. In all investigated breeds the rams with relatively high level of DNA methylation of gene SIRT1 in spermatozoa (50-68%) demonstrated a high conception rate at lambing (&gt; 70%). In conclusion, the DNA methylation level of the SIRT1 gene in ram spermatozoa is determined by both the breed and the age of the animals and correlates with fertilizing ability of sperm. Keywords: SIRT1 methylation, ram spermatozoa, Lacuane, East Friesian, Assaf breeds

Utjecaj hranidbe s dodatkom trutovskog legla na folikulogenezu u nazimica

The biological properties of bee drone brood make it an ideal additive for growth promotion in animal husbandry instead of banned hormonal anabolics and antibiotics. However, the drone brood action on mammalian ovaries has not been well studied. The present study analyzes the impact of drone brood homogenate (DBH) in the diet of growing gilts on folliculogenesis. Large White female pigs at the age of 35 days were randomly divided into two groups of 10 animals each, and fed with the same basal diets. The experimental group was supplemented with 25 mg/kg forage of DBH for 180 days, after which the animals were slaughtered and morphometric, histological and immunohistochemical evaluation of their ovaries was performed. In addition, the expression of ovarian growth factors BMP15 and GDF9 in oocytes and cumulus cells was analyzed by RT-PCR. A significant increase in body weight and average daily gain at day 145 in the DBH-supplemented group was established. The length of the ovaries in the treated animals also was enhanced. More pools of primordial follicles, involved in intensive growth, as well as a larger diameter of primary and tertiary follicles were found in the ovaries of DBH-supplemented animals. These findings corresponded with an increase in the expression of GDF9 mRNA in the oocytes and cumulus cells. At the same time, signs of atresia in the Graafian follicles of treated animals were observed. The supplementation with DBH stimulates the early stages of folliculogenesis in gilts, but provokes atresia in the last stage of follicular development.Biološka svojstva pčelinjeg trutovskog legla čine ga idealnim dodatkom za poboljšanje rasta u stočarstvu umjesto zabranjenih hormonskih anabolika i antibiotika. Ipak, utjecaj trutovskog legla na jajnike u sisavaca još uvijek nije dovoljno istražen. U ovom se radu analiziran je utjecaj homogenata trutovskog legla (DBH) u prehrani nazimica na folikulogenezu. Ženke pasmine veliki jorkšir u dobi od 35 dana nasumično su podijeljene u dvije skupine po 10 životinja, s jednakim osnovnim prehrambenim obrokom. Pokusnoj skupini u krmu je dodavano 25 mg/kg DBH-a tijekom 180 dana, nakon čega su životinje usmrćene te je učinjena morfometrijska, histološka i imunohistokemijska procjena jajnika. Zatim je u oocitama i stanicama kumulusa RT-PCR-om analiziran izražaj faktora rasta jajnika, BMP15 i GDF9. U skupini s dodatkom prehrani DBH ustanovljen je znakovit porast tjelesne mase i prosječnog dnevnog prinosa 145. dan. Povećana je i dužina jajnika u pokusnih životinja. U životinja hranjenih dodatkom pronađeno je više nakupina primordijalnih folikula uključenih u intenzivan rast, kao i veći promjer primarnih i tercijarnih folikula. Ovi su rezultati u skladu s porastom izražaja GDF9 mRNA u oocitama i stanicama kumulusa. Istodobno su opaženi znakovi atrezije u Graafovim folikulima pokusnih životinja. Dodatak prehrani DBH stimulira rane stadije folikulogeneze u nazimica, ali uzrokuje atreziju u posljednjem stadiju folikularnog razvoja

EffEct of brEEding sEason on thE kinEmatic paramEtErs and morphology of ram&apos; spErm from synthEtic population bulgarian milk shEEp brEEd

abstract AbADjievA, D., M. Chervenkov, r. StefAnov, n. MetoDiev, e. kiStAnovA, D. kAChevA and e. rAyChevA, 2014. effect of breeding season on the kinematic parameters and morphology of ram&apos; sperm from synthetic population bulgarian milk sheep breed. Bulg. J. Agric. Sci., the aim of this study was the investigation of the breeding season effect on the kinematic and main spermatological parameters of the rams from Synthetic Population Bulgarian Milk sheep breed (SPBM), new Bulgarian breed certificated in 2005. the experiment was carried out with seven rams. Two consecutive ejaculates from each ram were obtained by artificial vagina before and during the breeding campaign (n=28). overall sperm motility and the individual kinematic parameters of motile spermatozoa were assessed by the computer-aided sperm analysis system Sperm Class Analyzer (SCA). the sperm morphology was estimated after sperm blue stain and calculated as a percent of abnormal cells among 100 sperm cells from several fields on the slide. it was found that the ejaculates obtained from SPbM rams during the breeding season had better features of sperm motion kinetics. the values of the velocity parameters (P&lt; 0.05), motility (P&lt; 0.05), and percentages of spermatozoa with rapid (P&lt; 0.01) and medium (P&lt; 0.001) speed were higher than those from the ejaculates collected before the breeding season. Minor and not significant changes in the kinematic parameters of motile spermatozoa in consecutive ejaculates were observed. No significant differences were established in morphological status of spermatozoa in nonbreeding and breeding season. It seems that the better sperm motility kinematic parameters during the breeding season ensure the higher sperm fertility and success on the future insemination

Desmoglein autoantibodies and disease severity in pemphigus patients – correlations and discrepancies

Aim: To assess the correlation between the levels of anti-desmoglein-1 and anti-desmoglein-3 autoantibodies and disease severity in pemphigus patients. Materials and methods: Thirty-eight pemphigus patients aged 30 to 87 years were included in the study. All patients underwent clinical examination, pemphigus disease zone index assessment, histopathological and direct immunofluorescence tests, and assessment of desmoglein-1 and desmoglein-3 autoantibodies by enzyme-linked immunosorbent assay. Results: Twenty-eight out of 38 serum samples exceeded the cut-off value of anti-desmoglein-1, and 26 of 38 sera had positive anti-desmoglein-3 antibodies. One serum from 38 controls had positive anti-desmoglein-1 antibodies. Seven (18.4%) patients experienced a mild course of the disease, 16 (42.1%) patients experienced moderate, and 15 (39.5%) patients suffered from severe pemphigus. A significant correlation between disease severity and both autoantibody levels was observed, but there were exceptions. Conclusions: There is a significant correlation between anti-desmoglein antibodies and disease severity in the entire group, but there are also discrepancies in some cases

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA

Improving the reporting quality of intervention trials addressing the inter-individual variability in response to the consumption of plant bioactives: quality index and recommendations

PURPOSE: The quality of the study design and data reporting in human trials dealing with the inter-individual variability in response to the consumption of plant bioactives is, in general, low. There is a lack of recommendations supporting the scientific community on this topic. This study aimed at developing a quality index to assist the assessment of the reporting quality of intervention trials addressing the inter-individual variability in response to plant bioactive consumption. Recommendations for better designing and reporting studies were discussed. METHODS: The selection of the parameters used for the development of the quality index was carried out in agreement with the scientific community through a survey. Parameters were defined, grouped into categories, and scored for different quality levels. The applicability of the scoring system was tested in terms of consistency and effort, and its validity was assessed by comparison with a simultaneous evaluation by experts' criteria. RESULTS: The "POSITIVe quality index" included 11 reporting criteria grouped into four categories (Statistics, Reporting, Data presentation, and Individual data availability). It was supported by detailed definitions and guidance for their scoring. The quality index score was tested, and the index demonstrated to be valid, reliable, and responsive. CONCLUSIONS: The evaluation of the reporting quality of studies addressing inter-individual variability in response to plant bioactives highlighted the aspects requiring major improvements. Specific tools and recommendations favoring a complete and transparent reporting on inter-individual variability have been provided to support the scientific community on this field