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The maintenance of high vitality and motility of ram's spermatozoa during storage at low temperatures has a crucial role for successful fertilization. This study evaluates the effect of the natural antioxidant oregonin on ram semen quality, stored at 5 °C for 48 h. Еighteen ejaculates (three repetitions for 6 ejaculates) from three local breed rams, collecting by artificial vagina, with volume > 1 ml, concentration > 1 × 109/ml and mass motility >3.5 were used for chilling. Each ejaculate was separated in two equal parts, diluted with Tris-glucose-glycerol-egg yolk extender with no oregonin or supplemented with 100μМ oregonin until adjustment of the sperm concentration to 200 × 106 cells/ml and stored at 5 °C for 48 h. The semen quality assessment was based on the main kinematic (by CASA analysis), morphological parameters (by BrightVit kit staining) and mitochondrial status (by MitoView staining) of the spermatozoa on 0, 24 and 48 h of storage, and on in vivo fertility test. Oregonin did not impair the morphology and kept sustained motility of ram spermatozoa stored at 5 °C for 48 h. The curvilinear velocity indicated faster movement of the oregonin treated sperms that corresponded with high percent of spermatozoa with active mitochondria in these samples. The fertilizing capacity of spermatozoa was preserved and pregnancy rate in the experimental group was 80% versus 60% in control. In conclusion, this study provides a new data about positive effect of the natural antioxidant oregonin, supplemented to the extender, on chilled ram semen quality, including fertilizing ability.

14-th Scientific Conference of Bulgarian FP of EFSA - Book

The aim of this study was to evaluate the effect of ascorbic acid (Vitamin C) and 2-mercaptoethanol (ME) added to buffalo semen samples after thawing for 1 h and 2 h incubation. Ejaculates (n = 20) from eight Bulgarian Murrah buffaloes were frozen in pellets and straws at a final sperm concentration of 80 × 106 mL. We investigated the effect of supplementations on the motility, morphology of spermatozoa at 1 h and 2 h after thawing, sperm plasma membrane status and mitochondrial trans-membrane potential. The comparative CASA analysis of the motility and velocity parameters of spermatozoa showed significant differences in the progressive motility and in the VCL values in favour of control vs. samples with additives (p

International audience; Scope: Flavanols are important polyphenols of the human diet with extensive demonstrations of their beneficial effects on cardiometabolic health. They contribute to preserve health acting on a large range of cellular processes. The underlying mechanisms of action of flavanols are not fully understood but involve a nutrigenomic regulation. Methods and Results: To further capture how the intake of dietary flavanols results in the modulation of gene expression, nutrigenomics data in response to dietary flavanols obtained from animal models of cardiometabolic diseases have been collected and submitted to a bioinformatics analysis. This systematic analysis shows that dietary flavanols modulate a large range of genes mainly involved in endocrine function, fatty acid metabolism, and inflammation. Several regulators of the gene expression have been predicted and include transcription factors, miRNAs and epigenetic factors. Conclusion: This review highlights the complex and multilevel action of dietary flavanols contributing to their strong potential to preserve cardiometabolic health. The identification of the potential molecular mediators and of the flavanol metabolites driving the nutrigenomic response in the target organs is still a pending question which the answer will contribute to optimize the beneficial health effects of dietary bioactives.

In this study the individual profiles of seminal plasma proteins (SPP) in rams of three breeds – Merino, Pleven Blackhead and Ile de France - were analysed. The study was carried out with three rams at 3, 6 and 10 years of age, grown and fed under similar conditions. Eighteen ejaculates (6 ejaculates from each ram) were evaluated by Sperm Class Analyzer. The total SPP concentration was measured spectrophotometrically. The separation and characterisation of SPP was performed by HPLC and one dimensional polyacrylamide gel electrophoresis (SDS-PAGE). There were no significant differences between the characteristics of ejaculates and the main kinematic parameters of the sperm in the breeds studied. Chromatograms showed specific profiles with 9, 10 and 11 protein peaks for Merino, Pleven Blackhead and Ile de France breeds, respectively. The total SPP concentration was the highest in the Pleven Blackhead breed and the lowest in Ile de France breed. The major parts of SPP in the three breeds were identical. The seminal plasma of Merino breed contained proteins with molecular mass of 30.3 kDa, 15.7 kDa and 15.2 kDa that were not present in the other two breeds. In the Ile de France and Pleven Blackhead samples only, two proteins with molecular masses of 39.7 kDa and 21.1 kDa, were observed. In conclusion, the detection of specific proteins can be used as a biological marker for sheep breed identification.

Simple Summary Shortening the inter-calving interval is the most important problem in buffalo breeding, especially on organic farms, where the use of hormones is prohibited. The demand for herbal preparations that stimulate the activity of ovaries, as well as the choice of an appropriate time for their application, remains essential today. This study provides the results of the positive effect of the herbal product, which identified a wide range of bioactive substances—carotenoids, flavonoids, and fatty acids, including esters of trienoic fatty acids, on the resumption of the cyclic ovarian activity in early postpartum buffaloes. Overcoming postpartum anestrus in buffaloes is highly cost-effective due to cost savings for keeping dry, unproductive animals. Abstract This study evaluates the phytochemical composition and ability of herbal preparation AyuFertin, supplemented shortly after calving, to resume the cyclic ovarian activity in postpartum Bulgarian Murrah buffaloes. In total, 13 healthy Bulgarian Murrah buffaloes at the age of 4–10 years, calved in March–April 2019 were involved in the experiment. Seven experimental buffaloes were treated individually per os with AyuFertin according to producer instruction. All animals were subjected to regular ultrasound examination of ovaries. GC-MS analysis of fatty acids composition and HPLC-DAD analysis of carotenoid and tocopherol content in AyuFertin were conducted. The determination of estrus occurrence and natural mating were done by a fertile bull. The development of the large follicles (12.5–14.3 mm) in 85.7% of treated animals was observed on day 24 postpartum compared to 0% in controls. Clinical signs of estrus were recorded in 71.4% of the animals, followed by an 80% pregnancy rate versus 16% in controls within 70 days after calving. In conclusion, the supplementation of the bioactive herbal product AyuFertin from day 21 postpartum can stimulate the follicular growth in the buffalo’s ovary, which leads to the development of follicles with ovulatory capacity, followed by successful ovulation. The above-mentioned treatment resulted in a better pregnancy rate in the treated multiparous compared to the control buffaloes bred by natural service.

Despite the well-known health properties of microalga Arthrospira (Spirulina) platensis, little is known about its effects on female reproduction. The aim of this study was to establish whether including A. platensis in the diet during sexual maturation has an effect on the folliculogenesis and expression patterns of growth factors as bone morphogenetic protein 15 (BMP15) and growth differential factor 9 (GDF9) in the ovaries of the mothers and F1 female offspring in rabbits. The research was conducted using 28 white New Zealand rabbits in total, and seven were individually treated with 350 mg kg-1 of A. platensis at 2 to 6 months of age, prior to the insemination. The morphometric evaluation of the ovaries, RT-PCR, and immunohistochemical analyses of GDF9 and BMP15 expression in the oocytes and cumulus cells of both generations was performed. Arthrospira platensis supplementation led to an increase in the weight of ovaries, in the density of the primary and secondary follicles in the ovaries of treated mothers and their F1 offspring, and in the live body weight of offspring born to the treated mothers. BMP15 expression levels in the oocytes and cumulus cells, and GDF9 expression in the oocytes of treated mothers were enhanced. This increase in BMP15 expression levels was shown to be inherited in the oocytes and cumulus cells of the F1 females born to treated mothers. These data suggests that the A. platensis-supplemented diet during the sexual maturation exerts transgenerational beneficial effects on the folliculogenesis in rabbits.

The aim of the present investigation was to study the effect of Arthrospira (Spirulina) platensis supplemented diet on follicular development and related endocrine parameters, such as estradiol and progesterone levels as well as ghrelin levels in pre-pubertal gilts. Twenty-one 60-day-old Danube White gilts, randomly divided in three groups—control and two experimental—were involved in the experiment. The diet of experimental animals was supplemented with 2 and 3 g of dry biomass/capita daily of the micro alga A. platensis (SP), respectively, during 120 days. Pigs weighing between 109.3 and 114.7 kg were slaughtered at the age of 180 days. The visual macroscopic and histological analysis of removed ovaries was done. Progesterone and estradiol levels by radioimmunoassay (RIA) and ghrelin by ELISE methods were measured. The presence of corpora lutea in the ovaries of animals from all three groups as well as the progesterone and estradiol levels indicated that the animals were cycling and puberty had started at the time of slaughtering. In the SP 3-g group, the number of large follicles and corpora lutea was significantly higher compared to the control group and the corpora albicans were found. A positive correlation was observed between ghrelin and progesterone among all groups as well as between estradiol and ghrelin in the experimental groups independent on dosages of SP. Arthrospira platensis-supplemented diet provoked a successful follicular development and an earlier ovulation during sexual maturation of pigs between 60 and 180 days.

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.

Mesenchymal stem cells (MSC) have been considered the promising candidates for the regenerative and personalized medicine due to their self-renewal potential, multilineage differentiation and immunomodulatory capacity. Although these properties have encouraged profound MSC studies in recent years, the majority of research has been based on standard 2D culture utilization. The opportunity to resemble in vivo characteristics of cells native niche has been provided by implementation of 3D culturing models such as MSC spheroid formation assesed through cells self-assembling. In this review, we address the current literature on physical and biochemical features of 3D MSC spheroid microenvironment and their impact on MSC properties and behaviors. Starting with the reduction in the cells' dimensions and volume due to the changes in adhesion molecules expression and cytoskeletal proteins rearrangement resembling native conditions, through the microenvironment shifts in oxygen, nutrients and metabolites gradients and demands, we focus on distinctive and beneficial features of MSC in spheroids compared to cells cultured in 2D conditions. By summarizing the data for 3D MSC spheroids regarding cell survival, pluripotency, differentiation, immunomodulatory activities and potential to affect tumor cells growth we highlighted advantages and perspectives of MSC spheroids use in regenerative medicine. Further detailed analyses are needed to deepen our understanding of mechanisms responsible for modified MSC behavior in spheroids and to set future directions for MSC clinical application.

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. Electronic supplementary material The online version of this article (10.1007/s00394-019-02069-3) contains supplementary material, which is available to authorized users., Key messages The reporting quality of human studies on inter-individual variation in response to plant bioactives is generally low and should be significantly improved.There is no specific guidance for reporting studies on inter-individual variation in response to plant bioactives.The assessment of reporting quality using a scale approach is considered a valuable tool in assessing compliance with the recommendations in the submission phase or during the reviewing process. It also provides a quantitative measure of the quality of studies to be used in meta-analysis.Eleven reporting criteria were developed and supported by detailed definitions and guidance for their scoring.The POSITIVe quality index was tested and demonstrated to be valid, reliable, and responsive.The use of the quality index and its supporting explanatory material (dictionary) as a guide for researchers, peer-reviewers, and journal editors will foster further complete and transparent reporting of data on inter-individual variability.The criteria used in the quality index can serve as additional guidance to inform the design and conduction of further studies on inter-individual variations in response to plant bioactives.Better reporting is expected to lead to a better understanding of the mechanisms and factors involved and thus better study designs with greater impact on policies and practice. Electronic supplementary material The online version of this article (10.1007/s00394-019-02069-3) contains supplementary material, which is available to authorized users.

The aim of this study was to test the effect of extenders containing different sugar in their composition on ram sperm motility and pregnancy rate of ewe’s following artificial insemination. Semen were collected from ten North-east Bulgarian fine-fleece breed and tested for quality. Semen was diluted with different extenders, with di- and trisaccharides. A series of experiments were repeated in triplicate. Total motility was determined by using Sperm Analysis (SCA, Microptic, Spain). A total of 200 North-east Bulgarian fine-fleece breed mature ewes were used for cervical insemination with a sperm dose at the concentration of 100 × 106 spermatozoa. Pregnancies were diagnosed 60 days after AI by - a real-time ultrasonic scan device (Alloka SSD 500). In conclusion, our experiments demonstrated that higher sperm motility after storage at 4°C for 24 hours and 48 hours has a ram spermatozoa diluted with extender 1, with combination of disaccharides (sucrose and lactose) and trisaccharides (rafinosa). This semen extender (number 1) can be used for successful insemination of ewes and to enhance pregnancy rate after artificial insemination.

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.

Selenium is a microelement of big importance for male reproduction. As a part of the antioxidative enzyme gluthationperoxidase and structural Se proteins, it plays pivotal role in the defense of spermatozoa against generated ROS and in ensuring of its motility. During the last years the interests to the organic forms of selenium was enhanced because of its better biological utilization. The present work aimed to study the effect of injective application of organic compound selenopyran in rams on the distribution of selenium content in blood and sperm and on the changes in sperm quality. The experiment was conducted with 5 rams from the Synthetic Population Bulgarian Milk breed at the age between 3-7 years and live weight 85-90 kg. The animals were injected once with an oil solution of the selenopyran in dose of 0.1mg/kg live weight (selenium content 24%) 45 days before starting the breeding season. The blood was collected before treatment and 45 days thereafter. At the same time the first and second ejaculates of rams were collected using artificial vagina and analyzed by Sperm Class Analyzer. The selenium content was measured in plasma, blood cells and sperm by atomic absorption spectrometry using SpectrAA 55B double beam spectrometer (Varian, Inc.). The results showed that one injection of organic compound of selenium - selenopyran in dose of 0.1mg/kg live weight ensured the increase and support of high level of selenium in blood (plasma and blood cells) and in sperm during investigated period. That lead to the proper spermatogenesis in testis and allowed the production of qualitative ejaculates with higher number of total spermatozoa as well as of spermatozoa with progressive motility.

Selenium is an essential micro-element in animal diet due to its high antioxidative properties. As a part of selenocystein it is an important constituent of the glutathione peroxidase (GPx) enzyme, which has a big importance for cell protection from oxidative damage. The aim of the present work was the investigation of the selenopyran effect on the antioxidative state of the pig ovary. The experiment was conducted with 18 gilts of Danube white breed randomly divided into two groups between 120 - 228 days of age. The animals received equal basal diets without selenium additives. The experimental gilts were injected once per month intramuscularly with oil solution of preparation selenopyran (9-phenylsymmetrical octahydroselenoxanthene) ensured 0.1 mgSe/kg live weight. After slaughtering, the ovaries were used for histological analysis and estimation of the selenium content in ovarian tissue by the atomic absorption spectroscopy method. The GPx activity in ovary homogenates using the colorimetric assay kit (BioVision) was measured. The expression of γ-glutamyl transpeptidase (GGT) in ovaries by immunochistochemical method was estimated. The selenopyran treatment leads to significant (P0.05) in ovarian tissue. Enhancement of GPx activity in the ovaries of experimental group was observed (142.61±6.6 versus 122.28±3.4U/gP, P

Crude fibers (CF) take a special place in the growing rabbit`s nutrition. In depend on their origin and composition, CF have digestibility 17- 25% in the roughages and up to 40-50% in the green and concentrate feeds. They are very important in the regulation of the processes of both digestibility and bacterial synthesis. The optimal CF quantity for growing rabbits is 12-15%. Alfalfa hay is preferred source of CF for rabbits, but it is too expensive. But, there are other sources of CF which are more suitable because of their price are lower. The aim of the present study was to investigate the effect of different sources of CF in the mixed rations for rabbits on blood biochemical changes, Volatile Fatty Acids (VFA) production in the ceacum and some fermentative parameters. In this respect we have interest to study the next sources of crude fibers: dry distillers` grain of wheat (DDGSw), produced by Bulgarian firm as a source except for crude fiber - 13% and crude protein (CP) also - 32%; meadow hay and as well as wheat straw as main and chipper sources of CF in the growing rabbits diets. Three compound feeds were prepared. All compound feeds contained 20% oats, 15% barley, 16.40%wheat bran, 10% soybean meal, 5% sunflower meal. The main sources of CF for the diets were: 30% Alfalfa hay (control group); 10% DDGSw + 20% meadow hay (Experimental group I, EG I) and 15% DDGSw + 15% wheat straw (Experimental group II, EG II). The obtained results showed that there were no any incidences of digestive disorders in all investigated groups. Improvement of cecal fermentation led to better NGR and FOM. We made conclusion that DDGSw in combine with meadow hay or wheat straw could use as alternative sources of CF for growing rabbits.

The paper analyzes the scientific data included the author's own results about the opportunities to increase the reproductive potential of female animals by application of the natural no hormonal substances and the supposed mechanisms of their action on the reproductive system. Also the new scientific data related to the discovering of the "metabolic" hormones (leptin, ghrelin) and kisspeptin/GPR54 system as well as their relationship with metabolism and reproductive function in animals is discussed.

The effect of Bulgarian product Vemoherb-T (dry extract of Tribulus terrestris) on some characteristics of the reproductive capacity of Guinea fowl (Numida meleagris) was studied. A trial was conducted with 30 Pearl-gray Guinea fowls, at the age of 32 weeks, divided in two groups, 12 female and 3 male each. The birds from the two groups were fed the same mixture. The experimental group received the tested product once daily in a dose 10 mg/kg body weight for 12 weeks. The laying intensity, egg morphology and fertility, the weight of testes and ovaries were controlled. It was found a higher laying intensity (P

Pike are an important species and declines are a concern, because the species provides predatory balance to many lake ecosystems. The aim of the current study was to examine the testicular morphology and sperm parameters of the freshwater fish Esox lucius (pike). The present study was carried out on 18 male fish, species Esox lucius, which were captured from the beginning of September until of October, throughout the Danube River. Samples were processed for routine semen assessment by sperm class analyzer and light microscopy. Histological testis’ structure follows the general pattern described for other fishes. These descriptions are important for a better understanding of reproductive biology and phylogeny, for which data remain scarce in the literature. Middle sections of the testes were fully filled with spermatozoa different of other two parts as these results correspond with sperm motility. It can be concluded that recommended for insemination to use sperm from middle testicular part of Esox lucuis.

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