Plasma melatonin concentration during the early post-partum period in Thoroughbred mares and their foals.

The authors aimed to determine the plasma melatonin concentration in mares and their new-born foals in the early post-partum period. Blood samples were collected from the jugular vein of 53 mare-foal pairs within twelve hours after parturition. Plasma melatonin levels were measured by ELISA. The melatonin concentration, adjusted for the moment of parturition using a generalised linear model, was 34.58 pg mL-1 in mares. It was significantly lower (27.63 pg mL-1) in the new-born foals. However, the melatonin concentration declined differently by the end of the twelve hours, it decreased less in the offspring than in the mothers. An artificial light supplementation at the end of gestation reduced the melatonin concentration both in mares and their foals by about 10 pg mL-1, compared to the controls. An elevated melatonin production may be related to preparation of mares for parturition and ensures the chances of survival of offspring, therefore the melatonin may reach its peak at the moment of foaling regardless of its actual time. The effect of low melatonin concentration in new-born foals might be associated with the foal's health and subsequent performance. The need to monitor the melatonin concentration in the offspring justifies further studies.

Effect of pituitary adenylate cyclase-activating polypeptide supplementation, applied during or after vitrification on mouse embryo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 µM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 µM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

Dual Inhibitors of AChE and BACE-1 for Reducing Aß in Alzheimer's Disease: From In Silico to In Vivo.

Alzheimer's disease (AD) is a complex and widespread condition, still not fully understood and with no cure yet. Amyloid beta (Aß) peptide is suspected to be a major cause of AD, and therefore, simultaneously blocking its formation and aggregation by inhibition of the enzymes BACE-1 (ß-secretase) and AChE (acetylcholinesterase) by a single inhibitor may be an effective therapeutic approach, as compared to blocking one of these targets or by combining two drugs, one for each of these targets. We used our ISE algorithm to model each of the AChE peripheral site inhibitors and BACE-1 inhibitors, on the basis of published data, and constructed classification models for each. Subsequently, we screened large molecular databases with both models. Top scored molecules were docked into AChE and BACE-1 crystal structures, and 36 Molecules with the best weighted scores (based on ISE indexes and docking results) were sent for inhibition studies on the two enzymes. Two of them inhibited both AChE (IC50 between 4-7 µM) and BACE-1 (IC50 between 50-65 µM). Two additional molecules inhibited only AChE, and another two molecules inhibited only BACE-1. Preliminary testing of inhibition by F681-0222 (molecule 2) on APPswe/PS1dE9 transgenic mice shows a reduction in brain tissue of soluble Aß42.

Rapid Identification of Potential Drug Candidates from Multi-Million Compounds' Repositories. Combination of 2D Similarity Search with 3D Ligand/Structure Based Methods and In Vitro Screening.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach in early drug discovery. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compounds' databases. This approach can be combined with physico-chemical parameter and diversity filtering, bioisosteric replacements, and fragment-based approaches for performing a first round biological screening. Our objectives were to investigate the combination of 2D similarity search with various 3D ligand and structure-based methods for hit expansion and validation, in order to increase the hit rate and novelty. In the present account, six case studies are described and the efficiency of mixing is evaluated. While sequentially combined 2D/3D similarity approach increases the hit rate significantly, sequential combination of 2D similarity with pharmacophore model or 3D docking enriched the resulting focused library with novel chemotypes. Parallel integrated approaches allowed the comparison of the various 2D and 3D methods and revealed that 2D similarity-based and 3D ligand and structure-based techniques are often complementary, and their combinations represent a powerful synergy. Finally, the lessons we learnt including the advantages and pitfalls of the described approaches are discussed.

Practical experience with artificial insemination (AI) using fresh chilled and frozen semen in mares.

The objective of this study was to compare the efficiency of artificial insemination (AI) carried out with frozen and fresh, diluted and chilled semen under field conditions. One hundred and twenty-nine mares of different breeds were included in the study. Eighty-one out of the 107 mares inseminated with fresh, chilled semen got pregnant. Seven pregnant mares aborted and 74 foals were born. Out of the 22 mares inseminated with frozen semen, 17 mares got pregnant. Two mares out of the 17 pregnant mares aborted and finally 15 healthy foals were born. No difference was found between the two groups in the ratio of the foals born (P > 0.05). The comparison of medians for the number of insemination cycles did not show significant differences. However, a significant difference (Kruskal-Wallis test, P = 0.014) was found in the number of the inseminations per conception in favour of frozen semen (2.5 vs. 1.8 with fresh chilled and frozen semen, respectively). The Cox regression revealed that the type of semen has a significant impact (P < 0.001) on the service period (duration of the insemination period): the use of frozen semen prolonged the insemination period. This could be due to management issues, since re-insemination with frozen semen took place after only one/a few missed oestrous cycles not used for AI.

Family aggregation analysis shows a possible heritable background of equine grass sickness (dysautonomia) in a Hungarian stud population.

Equine grass sickness (also known as dysautonomia) is a life-threatening polyneuropathic disease affecting horses with approx. 80% mortality. Since its first description over a century ago, several factors, such as the phenotype, intestinal microbiome, environment, management and climate, have been supposed to be associated with the increased risk of dysautonomia. In this retrospective study, we examined the possible involvement of genetic factors. Medical and pedigree datasets regarding 1,233 horses with 49 affected animals born during a 23-year period were used in the analysis. Among the descendants of some stallions, the proportion of animals diagnosed with dysautonomia was unexpectedly high. Among males, the odds of dysautonomia were found to be higher, albeit not significantly, than among females. Significant familial clustering (genealogical index of familiality, P = 0.001) was observed among the affected animals. Further subgroups were identified with significant (P < 0.001) aggregation among close relatives using kinship-based methods. Our analysis, along with the slightly higher disease frequency in males, suggests that dysautonomia may have a genetic causal factor with an X-linked recessive inheritance pattern. This is the first study providing ancestry data and suggesting a heritable component in the likely multifactorial aetiology of the disease.

Design and Selection of Novel C1s Inhibitors by In Silico and In Vitro Approaches.

The complement system is associated with various diseases such as inflammation or auto-immune diseases. Complement-targeted drugs could provide novel therapeutic intervention against the above diseases. C1s, a serine protease, plays an important role in the CS and could be an attractive target since it blocks the system at an early stage of the complement cascade. Designing C1 inhibitors is particularly challenging since known inhibitors are restricted to a narrow bioactive chemical space in addition selectivity over other serine proteases is an important requirement. The typical architecture of a small molecule inhibitor of C1s contains an amidine (or guanidine) residue, however, the discovery of non-amidine inhibitors might have high value, particularly if novel chemotypes and/or compounds displaying improved selectivity are identified. We applied various virtual screening approaches to identify C1s focused libraries that lack the amidine/guanidine functionalities, then the in silico generated libraries were evaluated by in vitro biological assays. While 3D structure-based methods were not suitable for virtual screening of C1s inhibitors, and a 2D similarity search did not lead to novel chemotypes, pharmacophore model generation allowed us to identify two novel chemotypes with submicromolar activities. In three screening rounds we tested altogether 89 compounds and identified 20 hit compounds (<10 µM activities; overall hit rate: 22.5%). The highest activity determined was 12 nM (1,2,4-triazole), while for the newly identified chemotypes (1,3-benzoxazin-4-one and thieno[2,3-d][1,3]oxazin-4-one) it was 241 nM and 549 nM, respectively.

Inhibition of the LOX enzyme family members with old and new ligands. Selectivity analysis revisited.

Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.

Effects of T-2 and Fumonisin B1 combined treatment on in vitro mouse embryo development and blastocyst quality.

In the temperate region, most common mycotoxins are members of fusariotoxins. It often happens that food and forage are contaminated with two or more fusariotoxins at the same time. Effects of co-contamination are poorly documented, especially in the field of reproduction. The aim of our study was to assess the combined effect of the two common fusariotoxins, T-2 toxin (T-2) and Fumonisin B1 on early mouse embryo development in vitro. Embryo culture media contained either (1) 0.5 ng/ml T-2; 1, 2, or 10 ng/ml FB1 (group A, B, and C, respectively); or (2) 0.5 ng/ml T-2 and 1, 2, or 10 ng/ml FB1 (group TA, TB, and TC, respectively). Developmental rate, blastocoel expansion, cell number, and micronucleus rate were measured following 96 h culture. Although the developmental rate was similar to the control group (86.43% vs. 83.33, 78.79, 85.98, and 86.67%, respectively) in the case of single toxin treatments, the combined treatments induced significant decreases (14.5, 33.6, and 22.8% in TA, TB, and TC, respectively). The proportion of late blastocysts was lower in all treatments compared to control (83.6% vs. 0-83.6%). Combined treatment resulted in a significantly lower proportion of late blastocysts (25% in TA and 0% in TB and TC). Cell numbers decreased in all toxin-treated groups with a higher rate after combined treatments. No differences were detected in the micronucleus rate in the single or combined treatments compared to control. Our study shows that T-2 and FB1 toxins do not necessarily decrease the developmental rate, but co-contamination results in a significantly lower blastocyst rate and disturbs the blastocoel expansion as well. One possible explanation of this observation could be that the presence of two mycotoxins in the culture media intensifies their negative effects. All toxin treatments decreased the cell number in the blastocysts and this negative effect was more expressed after combined treatment.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

The impact of laser-assisted hatching on the outcome of frozen human embryo transfer cycles.

Biochemical modifications of zona pellucida (ZP) result in zona hardening. Zona hardening (ZH) is induced by several factors such as advancing maternal age, in vitro culture conditions and cryopreservation and adversely effects implantation. The objective of the clinical study was to determine whether or not laser-assisted hatching (LAH) applied on day 3 frozen embryos improves the outcome of frozen embryo transfer (FET) cycles in patients with recurrent implantation failure and/or advanced female age. In total, 413 patients of different ages with recurrent implantation failure (maximum three cycles) were involved into the study. Patients were allocated randomly into LAH and control groups. On the day of FET, after thawing and just before FET, the ZP was thinned using a laser system. In the control group no treatment was applied on frozen embryo before transfer. The main outcome measures were clinical pregnancy rate. Overall, the results indicate a tendency that LAH increased (P = 0.08) clinical pregnancy. However, for patients older than 37 years, LAH increased pregnancy rates significantly (P = 0.03). In the LAH and control groups, the age of patients and the number of transferred embryos influenced pregnancy rates (P = 0.01). For patients older than 37 years, no effect of number of transferred embryos was detected (P = 0.14). The incidence of multiple pregnancies also increased in the LAH group (P = 0.01). In conclusion, in older woman, to overcome the negative effect of zona hardening, LAH could be performed on frozen embryos as a routine strategy before FET in frozen cycles in order to increase the possibility of pregnancy formation.

Effects of T-2 mycotoxin on in vitro development and chromatin status of mouse embryos in preimplantation stages.

T-2 toxin is a mycotoxin produced by phytopathogenic fungi of the Fusarium genus and has many well-studied deleterious effects on mammalian cells and reproductive tract. Despite the wide scale studies, the effects on preimplantation stage embryos are lacking. The aim of our study was to investigate the impact of T-2 on the cleavage stage of mouse embryos with regard to development to blastocysts and nuclear chromatin status.Six-weeks-old BDF1 female mice were superovulated and placed together overnight with mature males. Zygotes were flushed 20 h after human chorionic gonadotropin injection and divided randomly into treated (supplemented with 0.5, 0.75, and 1 ng/ml T-2) and nontreated (control) groups. Embryos were cultured in vitro for 96 h. Developmental stage was evaluated in the 72(nd)- and 96(th)-h for assessment of development dynamics. At the end of culture period, blastocysts from treated and control groups with normal morphology were selected for nuclear chromatin analysis. Blastocysts were categorized (grade A, B, and C) depending on the proportion of blasomeres with micronuclei and/or lobulated nuclei.Our data show significant decrease in the proportions of blastocysts in the 0.75 and 1 ng/ml toxin-supplemented groups compared with the control group. Blastocyst rate did not differ in embryos treated with 0.5 ng/ml T-2 but 24 h delay was found in blastocoel formation in all the treated groups. Only grade A (21.1%) and B (78.9%) blastocysts were found in low-toxin-contaminated group similar to the control ones (50-50%). Grade C embryos appeared in the 0.75 ng/ml (10%) treated group and the rate increased significantly (33.3%) in the highest contaminated group.T-2 mycotoxin has a harmful effect on early embryo development which results in decreased blastocyst proportion, delayed blastulation, and increased rate of chromatin damage.

T-2 mycotoxin slows down the development of mouse blastocysts, decreases their blastomere number and increases chromatin damage.

The mycotoxin T-2 has many harmful effects on mammalian cells and reproductive functions. In the present study, the in vitro effect of T-2 toxin on mouse blastocysts was examined. Embryos were cultured in media supplemented with 0.5, 0.75 and 1 ng/ml T-2. Different exposure times were applied [96 h (treatment I) or 24 h following 72 h in toxin-free media (treatment II)]. Blastomere number, nuclear chromatin status and blastocoel formation were investigated in blastocysts. Our data show that the effect of T-2 toxin may vary depending on the stage of the embryo at the start of exposure. At 96 h of exposure, the blastocysts had blastomeres with normal chromatin quality but their developmental potential was decreased. After 24 h of exposure applied following a 72-h culture, blastomeres had a higher level of chromatin damage, although their developmental potential was the same as in the control embryos. In both cases, decreased mitotic rate was found, which resulted in decreased blastomere number even at low toxin concentration.

Involuntary reduction in vigour of calves born from sexed semen.

The objective of this study was to compare the reproductive traits of heifers and the development characteristics of their calves following artificial insemination (AI) with sexed and non-sexed semen. The analysed characteristics included conception rate, gestation length, calf birth weight, calf vigour, stillbirth rate, and twinning rate. Data of 530 calves produced with sexed and 1,163 calves produced with non-sexed semen were analysed. The General Linear Model (GLM) was applied to assess the influence of semen type, farm, season of insemination, the calf's sex and the inseminating sire on gestation length and calf birth weight. With the exception of gestation length (P > 0.05), all other traits studied were significantly (P < 0.01) influenced by the type of semen. The conception rate was 55% for conventional and 44% for sexed semen, and the average gestation length was 274.6 and 274.9 days, respectively. The mean calf birth weight was 37.47 kg for non-sexed and 36.75 kg for sexed semen. The stillbirth rate was 6.19% for conventional and 7.54% for sexed semen, while the twinning rate was 3.78% for conventional and 1.13% for sexed semen. The calves produced with non-sexed and sexed semen differed significantly in viability (P < 0.001), the latter having a lower calf vigour score. The use of conventional semen did not affect the ratio of female and male calves (52.7:47.3%; P > 0.05); however, artificial insemination with X-sorted sexed semen significantly altered the sex ratio of calves (85.1:14.9%, P < 0.01). The results obtained in this investigation are in agreement with the majority of studies which compared the fertility traits, sex ratio and calf characteristics depending on the application of artificial insemination with sexed or conventional semen.

Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

BACKGROUND: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts. METHODS: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups. RESULTS: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status. CONCLUSIONS: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

Correlation between bull fertility and sperm cell velocity parameters generated by computer-assisted semen analysis.

Motility is one of the most important characteristics associated with the fertilising ability of spermatozoa indicating their viability and structural integrity. Therefore, the examination of motility constitutes an integral part of semen analysis. Computer-assisted semen analysis (CASA) allows an accurate and objective assessment of different sperm motion characteristics with high repeatability. The aim of this study was to evaluate the different kinematic (velocity) parameters of frozen/thawed bull semen and determine if any of them could be correlated with their fertilising capability after insemination based on the achieved pregnancy rate. Ejaculates from 10 bulls were collected and frozen. The kinematic/velocity parameters of spermatozoa were measured by CASA and compared to the pregnancy results of almost 9,000 females artificially inseminated (AI) with frozen semen of any of the 10 tested bulls. The data of the experiments are summarised mainly with a focus on the effects of individual velocities (curvilinear velocity: VCL, straight-line velocity: VSL, average path velocity: VAP) on fertility rather than on the influence of progressive motility as a whole. We conclude that VAP is the most useful semen motility characteristic which has clinical relevance in the prediction of fertility.

Combination of 2D/3D ligand-based similarity search in rapid virtual screening from multimillion compound repositories. Selection and biological evaluation of potential PDE4 and PDE5 inhibitors.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost effective approach. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compound databases but the generated library requires further focusing by various 2D/3D chemoinformatics tools. We report here a combination of the 2D approach with a ligand-based 3D method (Screen3D) which applies flexible matching to align reference and target compounds in a dynamic manner and thus to assess their structural and conformational similarity. In the first case study we compared the 2D and 3D similarity scores on an existing dataset derived from the biological evaluation of a PDE5 focused library. Based on the obtained similarity metrices a fusion score was proposed. The fusion score was applied to refine the 2D similarity search in a second case study where we aimed at selecting and evaluating a PDE4B focused library. The application of this fused 2D/3D similarity measure led to an increase of the hit rate from 8.5% (1st round, 47% inhibition at 10 µM) to 28.5% (2nd round at 50% inhibition at 10 µM) and the best two hits had 53 nM inhibitory activities.

Pregnancy Rates of Holstein Friesian Cows with Cavitary or Compact Corpus Luteum.

Cavitary corpora lutea are commonly observed during the estrous cycle in bovines. Since the quality of the corpus luteum (CL) is extremely important during embryo transfer when embryos are implanted into the recipient, the ultrasonographic examination of the CL is becoming more and more important in terms of the outcome of the procedure. In the present study, a total of 2477 ultrasonographic transrectal diagnoses were performed, and data were collected between the years of 2018 and 2020 in a large-scale Holstein Friesian dairy farm in Hungary. In 91.1% (n = 2257) and in 8.9% (n = 220) of the cases, compact CLs and cavitary CLs, respectively, were diagnosed at pregnancy diagnosis. The presence of a cavitary CL on the ovary at pregnancy diagnosis increased the odds of remaining open after pregnancy by 21 times compared to the presence of a compact CL (OR = 21.0, p < 0.001) in the cows. The presence of cavitary CL was not influenced either by month or season. Ovarian cysts were detected in 196 cases (8.0%) in the examined animals. The presence of a cavitary CL decreased by 9 times when an ovarian cyst was also diagnosed (OR = 9.0, 1.6% vs. 9.5%, p < 0.001). The presence of an ovarian cyst decreased the odds of established pregnancy by 81 times (OR = 81.1, p < 0.001). Based on our results, the presence of a cavitary CL between days 31 and 42 after artificial insemination is associated with a smaller chance of conception in Holstein Friesian cows. The presence of an ovarian cyst decreases the occurrence of cavitary CL and the chance of conception.

Vitrification preserves chromatin integrity, bioenergy potential and oxidative parameters in mouse embryos.

BACKGROUND: The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos. METHODS: In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups. RESULTS: Vitrification induced low grade blastomere cytofragmentation (P < 0.05) and low chromatin damage only in embryos at the morula stage (P < 0.001). Mitochondrial (mt) distribution pattern was affected by vitrification only in early embryos (P < 0.001). Mitochondrial activity did not change upon vitrification in morula-stage embryos but it was reduced in blastocyst-stage embryos (P < 0.05). Intracellular ROS levels significantly increased in embryos at the morula and blastocyst stages (P < 0.001). Colocalization of active mitochondria and ROS increased only in vitrified blastocysts. CONCLUSIONS: In conclusion, this study elucidates the developmentally-related and mild effects of vitrification on morphology, nuclear and bioenergy/oxidative parameters of mouse embryos and demonstrates that vitrification is a suitable method for preserving predictive parameters of embryo ability to induce a full-term pregnancy.

[Selection of a melanine concentrating hormone receptor-1 (MCHR1) antagonists' focused library and its biological screening with AequoScreen]. / Melanin koncentráló hormon receptor-1 (MCHR1) antagonista fókuszált könyvtár kiválasztása és in vitro biológiai szurése AequosCreen esszével.

Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.