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. 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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. 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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). 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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 & 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 < 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 < 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 (> 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

PELE Fragmentation Dynamics

An analytical model that describes the PELE fragmentation dynamics is presented and compared with experimental results from literature. The model accounts for strong shock effects and detailed interactions taking place between the filling - the inner core of the ammunition - and the target (including shock wave, expansion wave, free surface and interface interactions). This model is also compared with a simplified model that uses the acoustic approximation and neglects the different wave interactions, which was employed by previously published PELE analytical models. The results are compared quantitatively in terms of fragments' radial velocities and qualitatively in terms of number of fragments, their mass distribution and the fragmentation length of the jacket. It is shown that strong shock effetcs and detailed wave interactions must be accounted for in order to simulate accurately the pressure evolution in the filling, which is the origin of the fragmentation dynamics, especially for impact velocities beyond 1000 m/s

Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris

The impact of VHT on the changes in BMP15 and GDF9 expression in both generations of female rabbits (FC_VHT-M/ FC_VHT-F1).

<p>The impact of VHT on the changes in BMP15 and GDF9 expression in both generations of female rabbits (FC_VHT-M/ FC_VHT-F1).</p

Immunohistochemical staining for BMP15 in the rabbit ovaries of the mothers and F1- female offspring generations.

<p>Localization of the protein BMP15 in: (a) oocytes of the primordial and primary follicles in the VHT-treated mothers ovary; (b) granulosa cells of the tertiary follicle in the VHT- treated mothers ovary; (c) primordial and primary follicles—negative control of antibody; (d) oocytes of primordial and primary follicles in the ovary of F1 offspring born to treated mothers; (e) oocytes and granulosa cells of the antral follicle in the ovary of F1 offspring born to treated mothers; (f) mouse testis—positive control of antibody; (g) Original magnification: (a–f) × 20.</p

The dynamic of BMP15 protein expression in the oocytes and cumulus cells of the follicles at different stages of the development in rabbit ovaries.

<p>Control M–mothers generation; Control F1- female offspring born to the control mothers; VHT-M- VemoHerb-T treated mothers; VHT-F1- female offspring born to the treated mothers; 0-I–primordial and primary follicles; II-III–secondary and preantral follicles; IV–antral follicles. *—significant differences, p < 0.05. Protein expression was estimated by the program ImageJ based on the intensity of immunostaining reactions on the defined square. At least 5 slides per animal were analysed. Significantly intensive signals were detected in the oocytes from primordial and primary follicles in the ovaries of treated mothers as well as in the cumulus cells from the antral follicles of F1 females born to the treated mothers.</p