Assessment of residual plant DNA in bulk milk for Grana Padano PDO production by a metabarcoding approach.

The aim of this study was to evaluate the ability of DNA metabarcoding, by rbcl as barcode marker, to identify and classify the small traces of plant DNA isolated from raw milk used to produce Grana Padano (GP) cheese. GP is one of the most popular Italian PDO (Protected Designation of Origin) produced in Italy in accordance with the GP PDO specification rules that define which forage can be used for feeding cows. A total of 42 GP bulk tank milk samples were collected from 14 dairies located in the Grana Padano production area. For the taxonomic classification, a local database with the rbcL sequences available in NCBI on September 2020/March 2021 for the Italian flora was generated. A total of 8,399,591 reads were produced with an average of 204,868 per sample (range 37,002-408,724) resulting in 16, 31 and 28 dominant OTUs at family, genus and species level, respectively. The taxonomic analysis of plant species in milk samples identified 7 families, 14 genera and 14 species, the statistical analysis conducted using alpha and beta diversity approaches, did not highlight differences among the investigated samples. However, the milk samples are featured by a high plant variability and the lack of differences at multiple taxonomic levels could be due to the standardisation of the feed rationing, as requested by the GP rules. The results suggest that DNA metabarcoding is a valuable resource to explore plant DNA traces in a complex matrix such as milk.

Comparison of the Performance and Microbial Community Structure of Two Outdoor Pilot-Scale Photobioreactors Treating Digestate.

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 genetic heritage of Alpine local cattle breeds using genomic SNP data.

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.

Real-time qPCR is a powerful assay to estimate the 171 R/Q alleles at the PrP locus directly in a flock's raw milk: a comparison with the targeted next-generation sequencing.

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.

Differential gene expression in cumulus oocyte complexes collected by ovum pick up from repeat breeder and normally fertile Holstein Friesian heifers.

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 20kDa, 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.

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo.

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 (-0.5

In vitro fertilisation with frozen-thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR.

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 mug/ml at a temperature of 37 degrees 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<0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatability of sorting was obtained with frozen-thawed bull semen that was subsequently used for IVF giving rise to viable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flow cytometric efficiency.

Genetic control of conventional labeling through the bovine meat production chain by single nucleotide polymorphisms using real-time PCR.

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.

Survival motor neuron (SMN) polymorphism in relation to congenital arthrogryposis in two Piedmont calves (piemontese).

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-->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.