Maternal peri-conceptional undernourishment perturbs offspring sperm methylome.

The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring. Sperm DNA methylation, measured by Reduced Representation Bisulfite Sequencing (RRBS), identified Differentially Methylated Regions (DMR) in offspring that experienced in utero undernutrition, both in UND (244) and FA (240), compared with CTR. Gene ontology (GO) analysis identified DMRs in categories related to sperm function, therefore we investigated whether the fertilizing capacity of the semen from the three groups differed in an in vitro fertilization assay. Spermatozoa from the undernourished groups showed lower motility and sperm chromatin structure abnormalities, represented by a higher percentage of DNA fragmentation and an increased number of immature cells, compared with CTR. While good quality blastocysts were obtained from all three groups, the proportion of embryos reaching the blastocyst stage was reduced in the UND vs CTR, an effect partially rescued by the FA treatment. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the semen of the resulting offspring, the effects of which in next generation remain to be elucidated.

Epigenetic analysis of high and low motile sperm populations reveals methylation variation in satellite regions within the pericentromeric position and in genes functionally related to sperm DNA organization and maintenance in Bos taurus.

BACKGROUND: Sperm epigenetics is an emerging area of study supported by observations reporting that abnormal sperm DNA methylation patterns are associated with infertility. Here, we explore cytosine-guanine dinucleotides (CpGs) methylation in high (HM) and low motile (LM) Bos taurus sperm populations separated by Percoll gradient. HM and LM methylation patterns were investigated by bisulfite sequencing. RESULTS: Comparison between HM and LM sperm populations revealed that methylation variation affects genes involved in chromatin organization. CpG Islands (CGIs), were highly remodelled. A high proportion of CGIs was found to be methylated at low/intermediate level (20-60%) and associated to the repetitive element BTSAT4 satellite. The low/intermediate level of methylation in BTSAT4 was stably maintained in pericentric regions of chromosomes. BTSAT4 was hypomethylated in HM sperm populations. CONCLUSIONS: The characterization of the epigenome in HM and LM Bos taurus sperm populations provides a first step towards the understanding of the effect of methylation on sperm fertility. Methylation variation observed in HM and LM populations in genes associated to DNA structure remodelling as well as in a repetitive element in pericentric regions suggests that maintenance of chromosome structure through epigenetic regulation is probably crucial for correct sperm functionality.

Differential biodiversity responses between kingdoms (plants, fungi, bacteria and metazoa) along an Alpine succession gradient.

Biological diversities of multiple kingdoms potentially respond in similar ways to environmental changes. However, studies either compare details of microbial diversity across general vegetation or land use classes or relate details of plant community diversity with the extent of microbially governed soil processes, via physiological profiling. Here, we test the hypothesis of shared responses of plant and rhizosphere bacterial, fungal and metazoan biodiversities (especially across-habitat ß-diversity patterns) along a disturbance gradient encompassing grazed to abandoned Alpine pasture, on acid soil in the European Central Alps. Rhizosphere biological diversity was inferred from eDNA fractions specific to bacteria, fungi and metazoans from contrasting plant habitats indicative of different disturbance levels. We found that soil ß-diversity patterns were weakly correlated with plant diversity measures and similarly ordinated along an evident edaphic (pH, C:N, assimilable P) and disturbance gradient but, contrary to our hypothesis, did not demonstrate the same diversity patterns. While plant communities were well separated along the disturbance gradient, correlating with fungal diversity, the majority of bacterial taxa were shared between disturbance levels (75% of bacteria were ubiquitous, cf. 29% plant species). Metazoa exhibited an intermediate response, with communities at the lowest levels of disturbance partially overlapping. Thus, plant and soil biological diversities were only loosely dependent and did not exhibit strictly linked environmental responses. This probably reflects the different spatial scales of organisms (and their habitats) and capacity to invest resources in persistent multicellular tissues, suggesting that vegetation responses to environmental change are unreliable indicators of below-ground biodiversity responses.

Effect of cryopreservation and semen extender on extracellular vesicles isolated from bull semen.

Introduction: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility. Methods: EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition. Results: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender. Conclusion: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH).

BACKGROUND: Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. RESULTS: We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. CONCLUSION: We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.

Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage.

Introduction: Freeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage. Methods: This work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C. Results and discussion: The results demonstrated better structural stability, namely lipid composition and DNA integrity, in VDE spermatozoa than FD ones, with outcomes at RT storage comparable to 4°C. Likewise, in VDE the embryonic development was higher than in FD samples (12.8% vs. 8.7%, p < 0.001, respectively). Our findings indicated that in large mammals, it is important to consider dehydration-related changes in sperm polyunsaturated fatty acids coupled with DNA alterations, given their crucial role in embryonic development.

Variations of follicular fluid extracellular vesicles miRNAs content in relation to development stage and season in buffalo.

In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR < 0.05) between An and pO groups. A subset of the most significant DE-miRNAs between An and pO groups targets genes which function is related to the lipid and steroid metabolism, response to glucocorticoid and oestradiol stimulus. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and pO-FF-EVs, which regulate IL6 release and cellular adhesion, respectively. In conclusion, these results demonstrated that the miRNA cargo of buffalo FF-EVs varies in relation to both follicular development and season.

Dose-response effects of estrogenic mycotoxins (zearalenone, alpha- and beta-zearalenol) on motility, hyperactivation and the acrosome reaction of stallion sperm.

BACKGROUND: The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. METHODS: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters were evaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after staining with fluoroscein-conjugated peanut agglutinin. RESULTS: Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hours exposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenol reduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent with hyperactivation was stimulated according to the following rank of potency: alpha-zearalenol > 17beta-estradiol > zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-line velocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinear velocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of live acrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short, alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cells and simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction without altering motility. Conversely, zearalenone and 17beta-estradiol did not induce the acrosome reaction but induced hyperactive motility albeit to a different extent. CONCLUSIONS: Apparently, the mycotoxin zearalenone has 17beta-estradiol-like estrogenic activity that enables it to induce hyperactivated motility of equine sperm cells, whereas the zearalenol derivatives induce premature completion of the acrosome reaction and thereby adversely affect stallion sperm physiology. The alpha form of zearalenol still possessed the estrogenic ability to induce hyperactivated motility, whereas its beta stereo-isomere had lost this property.

A Combined Flow Cytometric Semen Analysis and miRNA Profiling as a Tool to Discriminate Between High- and Low-Fertility Bulls.

Predicting bull fertility is one of the main challenges for the dairy breeding industry and artificial insemination (AI) centers. Semen evaluation performed in the AI center is not fully reliable to determine the level of bull fertility. Spermatozoa are rich in active miRNA. Specific sperm-borne miRNAs can be linked to fertility. The aim of our study is to propose a combined flow cytometric analysis and miRNA profiling of semen bulls with different fertility to identify markers that can be potentially used for the prediction of field fertility. Sperm functions were analyzed in frozen-thawed semen doses (CG: control group) and high-quality sperm (HQS) fraction collected from bulls with different field fertility levels (estimated relative conception rate or ERCR) by using advanced techniques, such as the computer-assisted semen analysis system, flow cytometry, and small RNA-sequencing. Fertility groups differ for total and progressive motility and in the abnormality degree of the chromatin structure (P < 0.05). A backward, stepwise, multiple regression analysis was applied to define a model with high relation between in vivo (e.g., ERCR) and in vitro (i.e., semen quality and DE-miRNA) fertility data. The analysis produced two models that accounted for more than 78% of the variation of ERCR (CG: R 2 = 0.88; HQS: R 2 = 0.78), identifying a suitable combination of parameters useful to predict bull fertility. The predictive equation on CG samples included eight variables: four kinetic parameters and four DNA integrity indicators. For the HQS fraction, the predictive equation included five variables: three kinetic parameters and two DNA integrity indicators. A significant relationship was observed between real and predicted fertility in CG (R 2 = 0.88) and HQS fraction (R 2 = 0.82). We identified 15 differentially expressed miRNAs between high- and low-fertility bulls, nine of which are known (miR-2285n, miR-378, miR-423-3p, miR-191, miR-2904, miR-378c, miR-431, miR-486, miR-2478) while the remaining are novel. The multidimensional preference analysis model partially separates bulls according to their fertility, clustering three semen quality variable groups relative to motility, DNA integrity, and viability. A positive association between field fertility, semen quality parameters, and specific miRNAs was revealed. The integrated approach could provide a model for bull selection in AI centers, increasing the reproductive efficiency of livestock.

High biodiversity in a limited mountain area revealed in the traditional production of Historic Rebel cheese by an integrated microbiota-lipidomic approach.

Historic Rebel (HR) cheese is an Italian heritage cheese, produced from raw milk during the summer grazing period in the Alps. The aim of this work was (i) to characterize the cheese microbiota, by 16S rRNA gene amplicons sequencing, and the volatile and non-volatile lipophilic fraction, by Gas Chromatography and Dynamic Headspace Extraction-Gas Chromatography-Mass Spectrometry, and (ii) to evaluate their respective associations. HR cheese was dominated by Firmicutes phylum (99% of the entire abundance). The core microbiota was formed by Streptococcus, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus genera together representing 87.2-99.6% of the total abundance. The polyunsaturated fatty acids composition showed a high PUFA n-3, PUFA n-6 and CLA content, two fold higher than typical plain cheeses, positively correlated with pasture altitude. A complex volatilome was detected, dominated in terms of abundance by ketones, fatty acids and alcohols. Total terpene levels increased at higher altitudes, being the main terpenes compounds α-pinene, camphene and ß-pinene. The HR cheese showed a great diversity of bacterial taxa and lipophilic fractions among producers, despite belonging to a small alpine area, revealing a scarce cheese standardization and a chemical fingerprint of a typical mountain cheese produced during the grazing period. A deeper knowledge of the variability of HR cheese due to its composition in microbial community and volatile compounds will be appreciated, in particular, by elite consumers looking for niche products, adding economic value to farming in these alpine areas.

Sperm Nuclei Analysis and Nuclear Organization of a Fertile Boar-Pig Hybrid by 2D FISH on Both Total and Motile Sperm Fractions.

A wide range of mammalian hybrids has recently been found by chance or through population-screening programs, but studies about their fertilizing capacity remain scarce and incomplete. Most of them are assumed to be sterile due to meiotic arrest caused by the failure of chromosome pairings. In this study, we evaluated both sperm meiotic segregation, by 2D fluorescent in situ hybridization (FISH) analysis, and sperm quality (Sperm Chromatin Structure Assay) by flow cytometer in a fertile boar-pig hybrid (2n = 37,XY) originating from a Nero Siciliano pig breed (Sus scrofa domesticus) and a wild boar (Sus scrofa ferus). Spermatozoa were also separated by a dual-layer (75-60%) discontinuous Percoll gradient, resulting in two fractions with a significantly better overall quality in the motile sperm fraction. These data were confirmed by FISH analysis also, where the frequencies of spermatozoa with a regular chromosome composition were 27% in total sperm fraction and 64% in motile sperm fraction. We also evaluated the nuclear architecture in all counted spermatozoa, showing a chromatin distribution changing when chromosome abnormalities occur. Our results demonstrate that the chromosome pairing has a minimal effect on the sperm segregation and semen quality of a boar-pig hybrid, making it fertile and harmful for the conservation of autochthonous pig breeds.

Investigating Genetic and Phenotypic Variability of Queen Bees: Morphological and Reproductive Traits.

The quality of the honeybee queen has an important effect on a colony's development, productivity, and survival. Queen failure or loss is considered a leading cause for colonies' mortality worldwide. The queen's quality, resulting from her genetic background, developmental conditions, mating success, and environment, can be assessed by some morphological measures. The study aims to investigate variability for traits that could assess the quality of the queen. Related animals were enrolled in this study. Variance components were estimated fitting a mixed animal model to collected data. Heritabilities of body and tagmata weights ranged from 0.46 to 0.54, whereas lower estimates were found for the tagmata width and wing length. Heritabilities estimated for the spermatheca diameter and volume, number of ovarioles, and number of sperms were 0.17, 0.88, 0.70, and 0.57, respectively. Many phenotypic correlations related to size were high and positive, while weak correlations were found between morphology and reproductive traits. Introducing a queen's traits in a selection program could improve colonies' survivability. Further research should focus on better defining the correlations between the individual qualities of a queen and her colony's performance.

In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity.

In this in vitro study the effects of mercuric chloride on the motility and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated using CASA method and Annexin analysis was used for detection of structural changes. The concentration of mercury in the medium varied from 5.0 to 83.3 microg HgCl(2)/mL. At Time 0 the highest motility was detected in the control group (67.09 +/- 8.72%). Motility in groups with mercury administration was lower in comparison with control. Significant differences were detected in groups with 50.0-83.3 microg HgCl(2)/mL (P < 0.001) at Time 0. After 60 and 120 minutes of incubation with mercuric chloride the motility significantly decreased almost in all experimental groups. Progressive motility had a decreasing trend in all experimental groups. At time 60 and 120 significant differences were noted in the group receiving 6.25-83.3 microg HgCl(2)/mL. Significant differences were detected in all experimental groups, except the group with the lowest mercuric chloride administration. The concentration-dependent decrease of spermatozoa progressive motility up to 50% of control was detected for groups receiving 50.0 - 83.3 microg HgCl(2)/mL at Time 0, for groups receiving 12.5-83.3 microg HgCl(2)/mL at Time 60 and 120, decreasing from 36.46 +/- 18.73% to 1.03 +/- 2.50%. Detailed evaluation of spermatozoa distance (DAP, DCL, and DSL) and velocity (VAP, VCL, and VSL) parameters as well as straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of spermatozoa revealed decrease in groups with the highest mercury concentration in comparison with the control group at all time periods. Detection of spermatozoa with disordered membrane was carried out for groups with higher mercury concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the mercury exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In mercury-exposed groups positive reaction proved alteration in anterial part of head (acrosome), connection part (connection piece) and in mitochondrial segment. Detected data evidently confirm adverse effects of high mercuric chloride concentrations in rabbit semen on spermatozoa motility parameters.

Amniotic microvesicles impact hatching and pregnancy percentages of in vitro bovine embryos and blastocyst microRNA expression versus in vivo controls.

Embryo development and implantation are dynamic processes, responsive to external signals, and can potentially be influenced by many environmental factors. The aims of this study were to evaluate the effects of a culture medium supplemented with amniotic-derived microvesicles (MVs) on in vitro embryo hatching after cryopreservation, and pregnancy rate following embryo transfer. In addition, miRNA profiling of blastocysts produced in vitro, with or without (control; CTR) amniotic MV supplementation, was also evaluated using blastocysts produced in vivo. In vitro embryos were cultured with and without amniotic MV supplementation. In vivo blastocysts were obtained from superovulated cows. Samples for RNA isolation were obtained from three pools of 10 embryos each (in vivo, in vitro-CTR and in vitro + MVs). Our results show that the hatching percentage of cryopreserved in vitro + MVs embryos is higher (P < 0.05) than in vitro-CTR embryos and the pregnancy rate with fresh and cryopreserved in vitro + MVs embryos is higher than in vitro-CTR embryos. In addition, the analysis of differently expressed (DE) microRNAs showed that embryos produced in vivo are clearly different from those produced in vitro. Moreover, in vitro-CTR and in vitro + MVs embryos differ significantly for expression of two miRNAs that were found in higher concentrations in in vitro-CTR embryos. Interestingly, these two miRNAs were also reported in degenerated bovine embryos compared to good quality blastocysts. In conclusion, MV addition during in vitro production of embryos seems to counteract the adverse effect of in vitro culture and partially modulate the expression of specific miRNAs involved in successful embryo implantation.

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature.

Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to - 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.

MicroRNAs of Equine Amniotic Mesenchymal Cell-derived Microvesicles and Their Involvement in Anti-inflammatory Processes.

Cell-derived microvesicles (MVs) are a recently discovered mechanism of cell-to-cell communication. Our previous data show that MVs secreted by equine amniotic mesenchymal-derived cells (AMCs) are involved in downregulation of proinflammatory genes in lipopolysaccharide-stressed equine tendon and endometrial cells. The aim of the present study was to evaluate whether AMC-MVs contain selected microRNAs (miRNAs) involved in inflammation. Two pools of cells, derived from 3 amniotic membranes each, and their respective MVs were collected. Small RNAs were extracted and deep sequenced, followed by miRNA in silico detection. The analysis identified 1,285 miRNAs, which were quantified both in AMCs and MVs. Among these miRNAs, 401 were classified as Equus caballus miRNAs, 257 were predicted by homology with other species (cow, sheep, and goat), and 627 were novel candidate miRNAs. Moreover, 146 miRNAs differentially expressed (DE) in AMCs and MVs were identified, 36 of which were known and the remaining were novel. Among the known DE miRNAs, 17 showed higher expression in MVs. Three of these were validated by real time polymerase chain reaction: eca-miR-26, eca-miR-146a, and eca-miR-223. Gene ontology analysis of validated targets showed that the DE miRNAs in cells and MVs could be involved both in immune system regulation by modulating interleukin signaling and in the inflammatory process. In conclusion, this study suggests a significant role of AMCs in modulating immune response through cell-cell communication via MV-shuttling miRNAs.

Different Culture Times Affect MicroRNA Cargo in Equine Amniotic Mesenchymal Cells and Their Microvesicles.

Conditioned medium (CM) and microvesicles (MVs) are produced using different protocols: CM is collected following 12-96 h of cell culture without renewal of tissue culture medium, while MVs are collected after overnight cell culture. For future comparative studies in regenerative medicine looking at the efficacy of CM and MVs, it is important to understand how the quality of cell secretions is affected by culture. The aim of this study was to evaluate whether the duration of culturing influences the micro-RNAs (miRNAs) cargo of equine amniotic mesenchymal cells (AMCs) and their MVs. The analysis identified 990 miRNAs. After one night, there were 347 differently expressed (DE)-miRNAs between MVs and cells, whereas after four nights there were 359. About 58.3% of the DE-miRNAs were shared between samples produced under the two conditions. The comparison between miRNA content in AMC cells cultured for one night versus four nights showed eight DE-Equus caballus (eca)-miRNAs, which target genes were involved in immune response to external stimulus, inflammatory response, and production of reactive oxygen species. Comparing MVs isolated from one or four nights, four DE-miRNAs that target genes regulating cell cycle progression and production of reactive oxygen species were found, but only eca-miR-214 was enriched in the MVs after four nights. In conclusion, after 4 days of cell culture, the profile of AMC miRNAs was altered, indicating a probable phenotypic transition versus a new cell culture environment and aging. After this time, MVs accumulated eca-miR-214, which may help cells survive or adapt to new culture conditions.

Effect of different number of frozen spermatozoa inseminated on the reproductive performance of rabbit does.

The relationship between the number of frozen spermatozoa inseminated and fertility rate and litter size at birth in rabbit does was investigated. Six hundred artificial inseminations (AI) were performed on multiparous lactating does with three spermatozoa concentrations: 10, 25, 50x10(6)spermatozoa/AI. All the does were synchronized with 20 UI of eCG 2 days before AI. The estimated sexual receptivity was 87%. The freezing-thawing procedure strongly reduced kinetic and functional traits (acrosome integrity, capacitation) of the spermatozoa. The number of spermatozoa inseminated did not affect the reproductive performance: the mean fertility rate and litter size values were 51.5% and 7.6%, respectively. Sexually receptive does (n=522) inseminated with frozen spermatozoa showed a 58.0% fertility rate whereas, non-receptive does (n=78), had a very poor fertility rate (7.8%).

Microvesicles secreted from equine amniotic-derived cells and their potential role in reducing inflammation in endometrial cells in an in-vitro model.

BACKGROUND: It is known that a paracrine mechanism exists between mesenchymal stem cells and target cells. This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication. METHODS: In this context, this study aims to understand the efficacy of MVs in in-vitro endometrial stressed cells in view of potential healing in in-vivo studies. For this purpose, the presence and type of MVs secreted by amniotic mesenchymal stem cells (AMCs) were investigated and the response of endometrial cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract the in vitro stress in endometrial cells induced by lipopolysaccharide was studied by measuring the rate of apoptosis and cell proliferation, the expression of some pro-inflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin 1ß (IL-1ß), and metalloproteinases (MMP) 1 and 13, and the release of some pro- or anti-inflammatory cytokines. RESULTS: MVs secreted by the AMCs ranged in size from 100 to 200 nm. The incorporation of MVs was gradual over time and peaked at 72 h. MVs reduced the apoptosis rate, increased cell proliferation values, downregulated pro-inflammatory gene expression, and decreased the secretion of pro-inflammatory cytokines. CONCLUSION: Our data suggest that some microRNAs could contribute to counteracting in-vivo inflammation of endometrial tissue.