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Background Assessment of genetic diversity and population structure provides important control metrics to avoid genetic erosion, inbreeding depression and crossbreeding between exotic and locally-adapted cattle breeds since these events can have deleterious consequences and eventually lead to extinction. Historically, the Alpine Arc represents an important pocket of cattle biodiversity with a large number of autochthonous breeds that provide a fundamental source of income for the entire regional economy. By using genotype data from medium-density single nucleotide polymorphism (SNP) arrays, we performed a genome-wide comparative study of 23 cattle populations from the Alpine Arc and three cosmopolitan breeds. Results After filtering, we obtained a final genotyping dataset consisting of 30,176 SNPs for 711 individuals. The local breeds showed high or intermediate values of genetic diversity compared to the highly selected cosmopolitan breeds. Patterns of genetic differentiation, multidimensional scaling, admixture analysis and the constructed phylogenetic tree showed convergence, which indicates the presence of gene flow among the breeds according to both geographic origin and historical background. Among the most differentiated breeds, we identified the modern Brown cattle. In spite of admixture events, several local breeds have preserved distinctive characteristics, which is probably due to differences in genetic origin and geographic location. Conclusions This study represents one of the most comprehensive genome-wide analysis of the Alpine cattle breeds to date. Using such a large dataset that includes the majority of the local breeds found in this region, allowed us to expand knowledge on the evaluation and status of Alpine cattle biodiversity. Our results indicate that although many of the analyzed local breeds are listed as endangered, they still harbor a large amount of genetic diversity, even when compared to some cosmopolitan breeds. This finding, together with the reconstruction of the phylogeny and the relationships between these Alpine Arc cattle breeds, provide crucial insights not only into the improvement of genetic stocks but also into the implementation of future conservation strategies.

This study aimed at examining and comparing the nutrient removal efficiency, biomass productivity and microbial community structure of two outdoor pilot-scale photobioreactors, namely a bubble column and a raceway pond, treating the liquid fraction of an agricultural digestate. Bacterial and eukaryotic communities were characterized using a metabarcoding approach and quantitative PCR. The abundance, composition, diversity, and dynamics of the main microbes were then correlated to the environmental conditions and operational parameters of the reactors. Both photobioreactors were dominated either by Chlorella sp. or Scenedesmus sp. in function of temperature, irradiance and the nitrogen compounds derived by nitrification. Other species, such as Chlamydomonas and Planktochlorella, were sporadically present, demonstrating that they have more specific niche requirement. Pseudomonas sp. always dominated the bacterial community in both reactors, except in summertime, when a bloom of Calothrix occurred in the raceway pond. In autumn, the worsening of the climate conditions decreased the microalgal growth, promoting predation by Vorticella sp. The study highlights the factors influencing the structure and dynamics of the microbial consortia and which ecological mechanisms are driving the microbial shifts and the consequent reactor performance. On these bases, control strategies could be defined to optimize the management of the microalgal-based technologies.

The hazard to human health represented by transmissible spongiform encephalopathies in sheep is one of the major reasons for implementing the genetic selection plan to break down prion diseases. The problem is particularly important because of the risk of disease transmission from ewe to lamb via milk or colostrum. In order to establish an active and convenient monitoring of the flocks already undergone genetic selection and thus, indirectly increase consumers’ security, the challenge of the work was quantifying the classical scrapie risk in bulk milk. A new quantitative real-time PCR assay for the estimation of the 171 R and Q allelic frequencies in a DNA pool representative of all the lactating ewes present in a flock was optimized and validated “in field”. The repeatability range was 3.69–5.27 for R and 4.20–5.75 for Q. The ruggedness of the allele frequencies resulted 4.26 for R and 4.77 for Q. Regarding the validation “in field”, none of the considered sources of variability (flock, month, number of genotyped animals and somatic cell count) showed a significant effect on flock and milk at the linear model. The targeted next-generation sequencing was also tested to evaluate its applicability in this context. Results show that the real-time PCR assay could represent a valid tool for the determination of 171 R/Q allele frequencies in bulk milk. The implementation of a service for breeder self-control along the production chain would aim to increase the production of high-security dairy products, while monitoring over time of the effects of genetic selection in the flocks.

Objective: To investigate the telomere length in bovine offspring produced by a cloned and control bull, and the telomerase activity in embryos produced with the same technology. Methods: Five daughters of a control and five daughters of a bull cloned using a fibroblast of the control were produced by IVF using sperm of the two bulls. Blood samples of the offspring were collected at 2, 6, and 12 months of age and the relative telomere length (RTL) was assessed by flow cytometry. At same time the body growth, hematological profile, and clinical biochemistry of the same progeny was extensively surveyed, and results have been reported in a previous work. Thereafter, the telomerase activity was assessed using a real time PCR quantitative assay in groups of embryos produced with the same technology. Results: The offspring of the clone exhibited a modest, but significant (P

The aim of this study was to establish whether perturbed gene expression during cumulus oocyte development causes repeat breeding in cattle. In this study, a repeat breeder was defined as a normal estrous cycling animal that did not become pregnant after three inseminations despite the absence of clinically detectable reproductive disorders. Transcripts of genes extracted from cumulus oocyte complexes (COC) that were collected from three repeat breeder and three normally fertile Holstein Friesian heifers were compared. Up to 40 COC were collected from each heifer by means of repeated sessions of ovum pick up in the absence of hormonal stimulation; immediately plunged into liquid nitrogen; and stored at −80 °C until analysis. For each heifer, RNA was extracted from the pooled COC and hybridized on GeneChip® Bovine Gene Array (Affymetrix). Analysis of gene expression profiles of repeat breeder and control COC showed that 178 genes were differentially expressed (log2 fold change > 1.5). Of these genes, 43 (24%) were up-regulated and 135 (76%) were down-regulated in repeat breeder relative to control heifers. This altered pattern of expression occurred in genes involved in several cellular biological processes and cellular components such as metabolism, angiogenesis, substrate/ion transport, regulation/signaling, cell adhesion and cytoskeleton. From these, 13 genes potentially involved in cumulus oocyte growth were subjected to validation by qRT-PCR and nine genes (annexin A1, ANXA1; lactoferrin, LTF; interferon stimulated exonuclease 20 kDa, ISG20/HEM45; oxidized low density lipoprotein receptor 1, OLR1; fatty acid desaturase 2, FADS2; glutathione S-transferase A2 and A4, GSTA2 and GSTA4; glutathione peroxidase 1, GPX1; endothelin receptor type A, EDNRA) were confirmed to be differentially expressed. This study identified potential marker genes for fertility in dairy cattle.

Despite many recent advances in single nucleotide polymorphism (SNP) genotyping technologies, there is still the need for methodologies with a reasonable throughput. In this study, we compared three PCR-based methods for SNP detection: (1) a conventional PCR-based allele detection system with fluorescent genotyping technology, (2) a SNaPshot methodology by single nucleotide primer extension and, (3) a real-time PCR-based method by allele-specific minor groove-binder probes. These three methodologies were used to analyze 104 meat samples of a particular Italian cattle breed known for producing excellent quality meat and for a characteristic increased development of muscle mass, caused by a point mutation (C313Y) in the Myostatin gene. The analysis revealed 98 samples to be homozygous (mh/mh) and five to be heterozygous (mh/+) for the mutation whereas one sample resulted to be homozygous for the wild type (+/+). The results obtained with the three different assays were consistent. Overall, all three methodologies proved to be efficient for allelic discrimination studies; however, real-time PCR was faster and allowed to genotype up to 96 samples in a single step, minimizing the number of steps required for samples manipulation.

Since January 2002, the European Union has adopted precise guidelines aimed at protecting the safety of meat and controlling the production chain. To this purpose, the conventional traceability of livestock and meat represents the main tool, but verification of traceability requires genetic support. At present, single nucleotide polymorphisms (SNPs) represent the most innovative molecular markers in genotyping studies. The aim of this study was to verify correct labeling in a bovine meat production chain by a real-time PCR protocol based on SNP analysis. Reference hair samples from 5,000 animals were randomly collected from 22 farms. Twelve hundred meat samples were collected at different steps of the bovine meat production chain. In particular, 1,000 meat samples were collected at the slaughterhouse and 200 samples from the same animals directly at the butcher's shop. The protocol was optimized and validated by testing a set of 16 SNP markers on 95 DNA samples from bovine sires of different breeds. Thereafter, the genotyping of 2,200 samples was conducted with a set of 12 selected SNPs to verify traceability of the meat production chain at three different stages: farm, slaughterhouse, and butcher's shop. Irregularities in conventional traceability were evidenced directly in 1.87% of the samples at the slaughterhouse. This percentage increased to 3.25% when sampling was conducted at the butcher's shop. This study demonstrates that despite the precautions adopted over the meat production chain, some critical points still exist that cause the loss of a correct association between registration numbers and samples.

Summary The objectives of the present work were to verify whether simultaneous exposure to Hoechst 33342 and UV irradiation during sorting by flow cytometry may induce gene point mutations in bovine sperm and to assess whether the dye incorporated in the sperm may imply a mutagenic effect during the embryonic development. To this aim, high-resolution melt analysis (HRMA) was used to discriminate variations of single nucleotides in sexed vs. non-sexed control samples. Three batches of sorted and non-sorted commercial semen of seven bulls (42 samples) were subjected to HRMA. A set of 139 genes located on all the chromosomes was selected, and 407 regions of the genome covering a total of 83 907 bases were analyzed. Thereafter, sperm of one sexed and one non-sexed batch of each bull was used in in vitro fertilization, and the derived embryos were analyzed (n = 560). One hundred and thirty-three regions of the bovine genome, located in 40 genes, were screened for a total coverage of 23 397 bases. The comparison between the frequencies of variations, with respect to the sequences deposited, observed in the sexed and non-sexed sperm (843 vs. 770) and embryos (246 vs. 212) showed no significant differences (P > 0.05), as measured by chi-square tests. It can be concluded that staining with Hoechst 33342 and exposure to UV during sorting does not lead to significant changes in the frequencies of variants in the commercial sexed semen and in embryos produced in vitro with the same treated sperm.

The gilthead seabream (Sparus aurata) is one of the main marine fish species cultured in Europe. In order to enhance the efficiency of aquaculture production, there is an increasing interest to develop selective breeding programs and molecular markers especially useful for tracing pedigrees without having to rear families separately. Here we describe 12 new polymorphic microsatellites loci, whose polymorphism was evaluated in a sample of 32 unrelated individuals. For each locus, the number of alleles vary from 4 to 23 alleles with mean expected and observed heterozygosity of 0.71 and 0.67, respectively. Two out of the 12 loci tested (SA5-E06 and SA3-G06) showed significant deviation from HWE after Bonferroni correction (P 0.05; adjusted P = 0.0008).These set of microsatellites could prove very effective for parentage inference and stock differentiation studies.

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR-1.5), medium (-0.5ER-NRR+0.5) and high (ER-NRR≥+1.5) fertility based on estimated relative non-return rates (ER-NRR), to the two categories of low and medium-high fertility. Heterospermic insemination with a Piedmontese reference bull was employed to define an index of competitive binding ability (CBI) to the zona pellucida using fluorochrome-labeled sperm, and a competitive fertility index (CFI) using embryo paternal assignment by single nucleotide polymorphism (SNP). Furthermore, kinetic parameters, membrane integrity and sperm DNA/chromatin integrity (% DFI) were assayed. Low fertility bulls had lesser (P0.05) values for total motility and membrane integrity, and a greater value of % DFI as compared to medium and high fertility groups. A modest (P0.001) correlation was reported among ER-NRR and total motility (r=0.30), progressive motility (r=0.26), membrane integrity (r=0.43) and % DFI (r=-0.26). While % DFI alone allowed the identification of 70% lowly fertile bulls, combining membrane integrity, average path velocity and CBI allowed for identification of 78% of the lowly fertile sires. Paternal assignment by SNPs resulted in 96% of successful assignments and could provide an alternative support to microsatellites for in vivo studies based on heterospermic fertilization for estimating fertility.

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen–thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen–thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen–thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 μg/ml at a temperature of 37 °C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91±2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P

International audience; The term arthrogryposis refers to a symptom complex that is characterised by congenital limb contractures. Arthrogryposis has been reported in man, in farm animals and in pets. Several forms have been reported to have a genetic origin in man. In Brown Swiss and Holstein Friesian cattle, congenital contractures have been recorded and classified as spinal muscular atrophy (SMA). The survival motor neuron gene (SMN) has been suggested as a candidate gene for SMA. In the last 20 years, the National Association of Piedmont Cattle have recorded arthrogryposis cases. We cloned and sequenced SMN cDNA extracted from the spinal cord samples of two animals: one Piedmont calf showing a severe clinical form of arthrogryposis and one normal Piedmont calf. In the affected calf, more than 50% of the 5$'$end clones showed a ATG $>$ TTG single nucleotide polymorphism (SNP) in exon 1 that should determine a Met $>$ Leu aminoacid change (single point mutation M3L). This mutation is associated with a 9 bp increase length of 5$'$UTR and to a TTC $\rightarrow$ TTT silent mutation in exon 1. No single point mutation or 5$'$end polymorphism was shown in healthy animals and in the remaining 50% of the clones from the affected calf. We hypothesise a possible pathogenic effect of the 5$'$end-exon 1 polymorphism.