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Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose–response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.

Made available in DSpace on 2021-06-25T11:18:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-28 Heat stress has a detrimental impact on cattle health, welfare and productivity by affecting gene expression, metabolism and immune response, but little is known on the epigenetic mechanisms mediating the effect of temperature at the cellular and organism level. In this study, we investigated genome-wide DNA methylation in blood samples collected from 5 bulls of the heat stress resilient Nellore breed and 5 bulls of the Angus that are more heat stress susceptible, exposed to the sun and high temperature-high humidity during the summer season of the Brazilian South-East region. The methylomes were analyzed during and after the exposure by Reduced Representation Bisulfite Sequencing, which provided genome-wide single-base resolution methylation profiles. Significant methylation changes between stressful and recovery periods were observed in 819 genes. Among these, 351 were only seen in Angus, 366 were specific to Nellore, and 102 showed significant changes in methylation patterns in both breeds. KEGG and Gene Ontology (GO) enrichment analyses showed that responses were breed-specific. Interestingly, in Nellore significant genes and pathways were mainly involved in stress responses and cellular defense and were under methylated during heat stress, whereas in Angus the response was less focused. These preliminary results suggest that heat challenge induces changes in methylation patterns in specific loci, which should be further scrutinized to assess their role in heat tolerance. Department of Animal Science Food and Nutrition – DIANA Nutrigenomics and Proteomics Research Centre – PRONUTRIGEN and Biodiversity and Ancient DNA Research Centre Università Cattolica del Sacro Cuore Istituto di Biologia e Biotecnologia Agraria Consiglio Nazionale delle Ricerche IBBA CNR School of Veterinary Medicine International Atomic Energy Agency Collaborating Centre on Animal Genomics and Bioinformatics Department of Production and Animal Health São Paulo State University (unesp) Department of Production and Animal Health São Paulo State University (unesp)

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P P

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: R2 = 0.88; HQS: R2 = 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 (R2 = 0.88) and HQS fraction (R2 = 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.

Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the oocyte molecular status in relation to season are poorly understood. This study characterizes the microRNA (miRNA) and transcriptomic profiles of oocytes (OOs) and corresponding follicular cells (FCs) from buffalo ovaries collected in the breeding (BS) and non-breeding (NBS) seasons. In the BS, cleavage and blastocyst rates are significantly higher compared to NBS. Thirteen miRNAs and two mRNAs showed differential expression (DE) in FCs between BS and NBS. DE-miRNAs target gene analysis uncovered pathways associated with transforming growth factor β (TGFβ) and circadian clock photoperiod. Oocytes cluster in function of season for their miRNA content, showing 13 DE-miRNAs between BS and NBS. Between the two seasons, 22 differentially expressed genes were also observed. Gene Ontology (GO) analysis of miRNA target genes and differentially expressed genes (DEGs) in OOs highlights pathways related to triglyceride and sterol biosynthesis and storage. Co-expression analysis of miRNAs and mRNAs revealed a positive correlation between miR-296-3p and genes related to metabolism and hormone regulation. In conclusion, season significantly affects female fertility in buffalo and impacts on oocyte transcriptomic of genes related to folliculogenesis and acquisition of oocyte competence.

Made available in DSpace on 2018-12-11T17:34:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Prototheca blaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T). Institute of Sciences of Food Production Italian National Research Council (CNR ISPA), Via Celoria 2 Institute of Agricultural Biology and Biotechnology Italian National Research Council (CNR IBBA), Via Einstein Council for Agricultural Research and Economics – Research Centre for Animal Production and Aquaculture (CREA-ZA), Via Lombardo 11 Department of Veterinary Hygiene and Public Health Universidade Estadual Paulista (UNESP) Department of Veterinary Medicine Federal University of Lavras Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER), Via Einstein Department of Veterinary Hygiene and Public Health Universidade Estadual Paulista (UNESP)

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.

Background Numerous studies have established a link between maternal diet and the physiological and metabolic phenotypes of their offspring. In previous studies in sheep, we demonstrated that different maternal diets altered the transcriptome of fetal tissues. However, the mechanisms underlying transcriptomic changes are poorly understood. DNA methylation is an epigenetic mark regulating transcription and is largely influenced by dietary components of the one-carbon cycle that generate the methyl group donor, SAM. Therefore, in the present study, we tested whether different maternal diets during pregnancy would alter the DNA methylation and gene expression patterns in fetal tissues. Results Pregnant ewes were randomly divided into two groups which received either hay or corn diet from mid-gestation (day 67 ± 5) until day 131 ± 1 when fetuses were collected by necropsy. A total of 1516 fetal longissimus dorsi (LD) tissues were used for DNA methylation analysis and gene expression profiling. Whole genome DNA methylation using methyl-binding domain enrichment analysis revealed 60 differentially methylated regions (DMRs) between hay and corn fetuses with 39 DMRs more highly methylated in the hay fetuses vs. 21 DMRs more highly methylated in the corn fetuses. Three DMRs (LPAR3, PLIN5-PLIN4, and the differential methylation of a novel lincRNA) were validated using bisulfite sequencing. These DMRs were associated with differential gene expression. Additionally, significant DNA methylation differences were found at the single CpG level. Integrative methylome and transcriptome analysis revealed an association between gene expression and inter−/intragenic methylated regions. Furthermore, intragenic DMRs were found to be associated with expression of neighboring genes. Conclusions The findings of this study imply that maternal diet from mid- to late-gestation can shape the epigenome and the transcriptome of fetal tissues, and putatively affect phenotypes of the lambs. Electronic supplementary material The online version of this article (10.1186/s12864-018-4509-0) contains supplementary material, which is available to authorized users.

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.

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (-12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of invitro-produced embryos, compared with those collected invivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived extracellular vesicles (EVs) improved their quality compared with control (CTR) (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671), and after cryopreservation, provided higher invitro embryo hatching and recipient pregnancy rate (Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). After these results, the aim of this study was to evaluate microRNA (miRNA) profiling of invitro-produced blastocysts with or without EV supplementation, using invivo-produced blastocysts as CTR. Invitro embryos were produced based on our protocol (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671) with or without 100×106 EVsmL−1 in synthetic oviductal fluid with amino acids (SOFaa) on Day 5 post-fertilisation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671). Grade 1 blastocysts (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. Invivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR, and vitro+EVs). Total RNA was isolated by a NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by an Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate

UNLABELLED Bovine mastitis caused by Prototheca spp. infection is increasing worldwide, therefore becoming more relevant to the dairy industry. Almost all Prototheca isolates from bovine mammary protothecosis came from P. zopfii genotype 2, with a lower prevalence of infection due to P. blaschkeae and rarely to P. wickerhamii. In this study, we report the development of two multiplex PCR assays able to discriminate among the three species responsible for bovine intramammary infection (IMI). Our assay is based on the specific amplification of new DNA target from mitochondria and chloroplasts partial sequences, of different Prototheca isolates. Both methods were set up using reference strains belonging to all Prototheca species and validated by the analysis of 93 isolates from bovine and buffalo IMI and bulk tank milk samples. The investigation involves 70 isolates from North, 13 from Central and 10 from South Italian regions. Isolates from bovine were most commonly identified as P. zopfii genotype 2, and only in one case as P. blaschkeae, whereas isolates from buffaloes belonged both to P. zopfii genotype 2 and P. wickerhamii. These findings proved the suitability of our multiplex PCRs as a rapid test to discriminate among pathogenic Prototheca strains. SIGNIFICANCE AND IMPACT OF THE STUDY This work reports PCR assays based on novel Prototheca spp. mitochondrial and chloroplastic target sequences. The multiplex PCR protocol described in this study is useful for rapid simultaneous detection of P. zopfii, P. wickerhamii and P. blaschkeae.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. Significance and Impact of the Study: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time. © 2017 The Society for Applied Microbiology

Background Small RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed. To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized. Results Sperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT). Conclusions Small RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3394-7) contains supplementary material, which is available to authorized users.

An autochthonous starter culture was used in the production of Protected Designation of Origin (PDO) Silter raw milk cheese, and compared to a control cheese in order to study its influence on microbial population dynamics during ripening. Curd and cheese at different ripening periods (0, 30, 60 and 200 days) from two independent dairies were analysed combining culture-based microbiological analysis, Length Heterogeneity-PCR and 16S microbiome profiling. The autochthonous starter determined higher lactic acid bacteria (LAB) levels for the first 30 days of ripening, without interfering with secondary microbiota that determines the tipicality of this cheese. Only a few genera and species persisted in the cheese despite the microbial richness of the curd. In addition, the high levels of different LAB reduced harmful microorganisms. The various analytic methods used resulted in discrepancies in the proportions of Enterococcus , Lactococcus and Lactobacillus spp., but the pivotal role of Streptococcus and Lactococcus genera was evident. The dominant species included those selected to formulate the starter ( St. thermophilus , Lc. lactis , Ln. mesenteroides ) as well as Lb. paracasei . The addition of autochthonous starter proved to be effective in controlling the first phases of the cheese-making, without compromising cheese typicality.

Whereas an organism’s genotype is relatively static throughout life, the epigenome is highly dynamic and can adapt, or be altered, in response to the external environment such as diet. Intrauterine exposure to nutrient availability can alter the establishment of epigenetic marks, not only in the exposed individuals, but also in their offspring. Inheritance of such an environmentally acquired phenotype by the subsequent generation occurs through epigenetic modifications in the germline. Here, using a genome-wide approach, we evaluate how modification of the maternal diet pre- (14 days) and post- (28 days) conception can affect methylation status of the sperm of male offspring. Specifically, using a sheep model, we focused on the effect of maternal undernutrition on adult sperm methylation and its long-term consequences on sperm physiology and quality. Moreover, we investigated if supplementation of folic acid, to increase the availability of methyl donors, could prevent or ameliorate the adverse uterine environment caused by maternal undernutrition. Male lambs obtained from mothers subjected to different nutritional regimens (UND: undernutrition; FA: undernutrition and folic acid supplementation) appeared normal at birth, with a comparable body weight until Day 30 postpartum. Sperm DNA methylation, obtained by reduced representation bisulfite sequencing, differed in offspring that experienced in utero undernutrition (UND and FA) compared with the control group (CTR). In particular, the number of differentially methylated regions (DMR) was lower when UND and FA groups were compared, whereas a higher number of DMR was observed by comparison of CTR with both experimental groups. In addition, a high percentage of DMR were shared between UND and FA groups when compared with CTR, clearly indicating a influence of maternal nutrition on the offspring sperm DNA methylation rearrangement. Gene ontology (GO) analysis showed variation in functional categories related to sperm functionality such as chondroitin sulfate synthesis, potassium ion import, and others related to metabolism (biotin and glucagon). Furthermore, using computer-assisted semen analysis and flow cytometric measurement, we observed lower a sperm motility index and higher incidence of chromatin structure alterations in spermatozoa collected from UND and FA groups compared with CTR. Finally, to verify the effect of such reported abnormality on lamb fertility, we used the semen for in vitro embryo production. While we obtained good quality blastocysts from all 3 groups, a reduction in the percentage of embryo development, partially compensated in the FA group, was found using spermatozoa from UND rams. Taken together, our results confirm that a nutritional stress during early mammalian development can lead to epigenetic modification in the offspring. This damage can be partially ameliorated with folic acid supplementation; however, some alteration still persists in the germline and could be passed to the next generation, with as yet unknown consequences.

Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation.

Reliable evaluations of soil biodiversity represent a key factor in understanding ecosystem services. To date, species-discriminating barcodes efficiently describe bacterial and fungal communities associated with environmental samples, whereas investigations of soil microfauna are often hampered by the lack of a marker region encompassing the taxonomic range of soil organisms. Two new PCR primer sets targeting the V4-V5 and V5-V7 variable regions of the ribosomal 18S RNA (18S rRNA) were designed to be specific for metazoans metabarcoding and capable of detecting the majority of their lineages. In silico and in vivo assays on four soil typologies were carried out to compare the newly developed primer sets with a selection of primers targeting the homologous gene, which were previously used to assess soil metazoan biodiversity. The new primer sets, both on the basis of the in silico and in vivo comparisons, were very selective and consistent when analysing metazoan biodiversity across the tested soil typologies. On the basis of the coverage index and taxonomic resolution, the new primers targeting the ribosomal 18S RNA outperformed the other primers, and they represent a promising tool for assessing soil metazoan biodiversity through metabarcoding approaches. biodiversity. The new primer sets, both on the basis of the in silico and in vivo comparisons, were very selective and consistent when analysing metazoan biodiversity across the tested soil typologies. On the basis of the coverage index and taxonomic resolution, the new primers targeting the ribosomal 18S RNA outperformed the other primers, and they represent a promising tool for assessing soil metazoan biodiversity through metabarcoding approaches.

International audience; This review, is a synopsis of advanced reproductive technologies in farm animals, including the discussion of their limiting factors as revealed by the study of offspring derived from embryos produced in vitro and through cloning. These studies show that the problems of epigenetic mis-programming, which were reported in the initial stages of assisted reproduction, still persist. The importance of whole-genome analyses, including the methylome and transcriptome, in improving embryo biotechnologies in farm animals, are discussed. Genome editing approaches for the improvement of economically-relevant traits in farm animals are also described. Efficient farm animal embryo biotechnologies, including cloning and the most recent technologies such as genome editing, will effectively complement the latest strategies to accelerate genetic improvement of farm animals.

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.

Coat pigmentation in birds and other vertebrates depends on the balance of two types of melanins: brown-grey-black eumelanin and yellow-red pheomelanin.1 Although it is known that more than 150 genes impact pigmentation, there are a few key genes in the melanogenic pathway: MC1R (melanocortin 1 receptor), ASIP (agouti signaling protein), MITF (microphthalmia-associated transcription factor), MLPH (melanophilin), TYRP1 (tyrosinase-related protein 1), SLC45A2 (solute carrier family 45 member 2) and TYR (tyrosinase).1-3 Previous studies have reported a correlation between genetic variations in the sex-linked TYRP1 gene and variation of plumage colour in Japanese quail, domestic pigeon and duck, but no data are available for falcons.4-6 Here, we investigated the involvement of polymorphisms of the TYRP1 gene in the brown plumage phenotype of Falco cherrug. Forty-two F. cherrug bred in captivity from a two-generation pedigree were evaluated to test the inheritance of a colour trait, i.e. wild-type and a lighter mutant (Fig. 1). The F1 generation consisted of 12 females, nine of which were light brown, and 18 males (Table S1). The sex-linked inheritance of the phenotype and the light brown plumage observed in the offspring of carrier males and wild-type females indicates a sex-linked inheritance of these mutations and the involvement of a Z-chromosome gene as previously described for other bird species (Fig. S1).7,8 The sequences of the TYRP1 exons were obtained from one wild-type and one mutant female by Sanger sequencing (Table S2). Exon 1 of the TYRP1 amplicon was then sequenced in all animals. Sequences were deposited in GenBank (Accession numbers KX181731 and KX181733). Two mutations - c.79C>T and c.79_80insT - were found in exon 1 of all light brown females. Translation predicts a frameshift change and a premature stop codon at position 81 of the deduced protein sequence, which in the wild-type form contains 536 amino acids. The cited-above mutation in the TYRP1 gene was found in all nine brown females of the F1 generation. Moreover, in three F0 and six F1 wild-type males double peaks downstream of the polymorphic site revealed that these animals were heterozygous for the mutations. Previous genetic studies on coat colour of mammalian and avian species also reported a key role of TYRP1 gene in coat or plumage color via the production of the dark eumelanin.

Mesenchymal stromal cells (MSC) were found to secrete many factors with therapeutic relevance for their antioxidants, antiapoptotic, antifibrotic, angiogenic, immunomodulatory, and chemiotactic activities. The culture supernatant of MSC (in our case, of equine amniotic-derived cells) is defined secretome or conditioned medium (CM) and it is composed of soluble and insoluble factors secreted by cells. Soluble factors are represented by cytokines and growth factors, whereas microvesicles (MV), recently demonstrated to be an integral component of cell-to-cell communication during tissue regeneration, represent insoluble factors (Bruno et al. 2009 J. Am. Soc. Nephrol. 20, 1053-1067). Our previous data showed that equine amniotic-derived CM administered in vivo in equine spontaneous tendon lesion is able to regenerate the injured tissue. overlapping the results obtained by using in vivo the cells of origin in the same pathology (Lange-Consiglio et al. 2013 Stem Cells Devel. 22, 3015-3024). We also studied the amniotic-derived MV and found that they are involved in down-regulation of pro-inflammatory genes in in vitro LPS-stressed equine tendon and endometrial cells (Lange-Consiglio et al. 2016 Stem Cells Devel. 25, 610-621; Perrini et al. 2016 Stem Cell Res. Ther. 7, 169). Usually, protocols to produce CM and MV are different: CM is collected after 48 to 96 h of culturing cells without renewal of tissue culture medium, whereas MV are collected after culturing cells overnight. In future comparative studies of regenerative medicine aiming to understand the efficacy of CM and MV, understanding the quality of secretion of cells according to culturing time is crucial. The aim of the present study was to evaluate whether different times of culture can influence the micro-RNA (miRNA) cargo of equine amniotic-derived cells and their MV. Biological triplicates of a pool of amniotic-derived cells from 2 amniotic membranes and their MV, collected after overnight or after 4 days, were used. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisSEqn 2000 (Illumina Inc., San Diego, CA, USA). Mirdeep2 on Illumina high-quality trimmed sequences was used to detect known miRNAs and to support the individuation of novel miRNAs. The analysis identified 335 Equus caballus miRNA, which were quantified in both amniotic cells and MV. Hierarchical clustering of 83 miRNA that were observed in all 12 samples clearly discriminated amniotic cells and MV. The profile of miRNA in AMC and MV was similar between overnight and 4 days of culture. Further functional studies on the predicted target genes and pathways involved in the biological effect of equine amniotic secretome will be performed.

Staphylococcus aureus is one of the most important causes of mastitis in dairy cattle. Based on previous research, Staph. aureus genotypes with different pathogenic and contagious properties can cause intramammary infection (IMI) and coexist in the same herd. Our study aimed to compare Staph. aureus strains from herds that differed in IMI prevalence using different molecular approaches such as ribosomal spacer (RS)-PCR, multilocus sequence typing (MLST), spa typing, ribotyping, pulsed-field gel electrophoresis (PFGE), and multiplex PCR. For this purpose, 31 dairy herds with Staph. aureus IMI were selected, and 16 of these were chosen for a comparison study: the 8 high-prevalence (HP) herds had Staph. aureus IMI prevalence >28% and the 8 low-prevalence (LP) herds had an IMI prevalence

Plant responses to herbivore insects involve direct and indirect defense with the production of signal molecules including jasmonic acid (JA) and its derivatives (e.g. methyl jasmonate, MeJA). In maize (Zea mays), root feeding by Diabrotica virgifera larvae activates an indirect defense mechanism, through enthomopathogenic nematodes that are recruited after Terpene Synthase 23 (tps23) upregulation and (E)-β-caryophyllene root emission. In order to gain insight into the correlation between JA signaling and response to Diabrotica attack, we analyzed tps23 expression and protein profiles in maize roots in response to MeJA treatment and insect infestation. Similar to herbivore feeding, MeJA treatment was found to increase tps23 transcript accumulation, with consistent variations for both treatments in maize lines differing in (E)-β-caryophyllene production. Analysis of root protein profiles showed specific alterations leading to the identification of three proteins that were induced by MeJA treatment. We focused on a peroxidase-like protein (Px-like) showing that the corresponding transcripts accumulated in all tested lines. Results show that exogenous application of MeJA upregulates tps23 expression and specifically alters protein patterns in maize roots. Parallel effects on tps23 transcript accumulation were observed upon hormone exposure and insect infestation in different maize lines. In contrast, Px-like transcript profiling showed differences between treatments. These results support the possible involvement of MeJA in mediating the upregulation of tps23 in response to Diabrotica attack.

Purpose S. aureus is the principal agent of bovine mastitis and it is known that the clinical outcome can vary in relation to the strains involved in the mammary infection. The aim of this work, funded by the Region Lombardia as "Mastfield" project, was to develop a diagnostic protocol for the identification of genetic subtypes of S. aureus responsible for bovine mastitis, with particular pathogenicity and infectivity. Methods S. aureus strains associated with intra-mammary infections were isolated from eight "case" (> 28% of infected cows) and eight "control" (< 4% of infected cows) herds. A total of 2015 cows were included in the study, milk samples were cultured with standard methods and the DNA was extracted from S. aureus strains using a protocol described in literature. A total of 650 strains isolated from single infected quarters were analyzed, 24 and 626 from "control" and "case" herds, respectively. All samples were analyzed both by a method based on the amplification of the 16S-23S rRNA intergenic spacer region (RS-PCR) and by multiplex PCR, in order to verify the presence of genes encoding for enterotoxins and other virulence genes. A subset of 55 strains representing the isolates from "control" and from "case" herds was also analyzed by ribotyping (automatic Riboprinter) and by protein profiling using MALDI-TOF MS. Results The RS-PCR analysis revealed 25 different profiles. In "case" herds, the analysis showed the presence of S. aureus belonging to one genetic profile spreading in the group. In 3 "case" herds the analysis showed more than one profile (maximum three different) one of which was in any case absolutely predominant. Moreover, in five out of the eight "case" herds, the RS-PCR analysis identified the genotype indicated in literature as characteristic of highly diffusive and pathogenic strains. Seven out of the eight "control" herds, revealed only one S. aureus profile, while in the eighth herd two different profiles circulated. Except for four strains, ribotyping gave a response similar to RS-PCR analysis in a cluster analysis. And more, MALDI-TOF analysis, proved to be able to group the S. aureus isolates with results close to RS-PCR. Finally, all the isolates from "case" herds possessed genes encoding for enterotoxins and showed genetic polymorphisms characteristic of pathogenic strains, while isolates from "control" herds were non-enterotoxigenic. Conclusions Our results demonstrated the validity of RS-PCR as a rapid tests for molecular typing of S. aureus strains isolated from bovine mastitis. The preliminary results of the ribotyping analisis and MALDI-TOF techniques showed that they could also used in molecular profiling of the strains, increasing the specificity of the analysis. Theese data could be usefull for the selection of a restricted panel of markers of virulence and diffusibility of the S. aureus strains isolated in the field.