Analysis of miRNAs in milk of four livestock species.

BACKGROUND: Milk is essential for mammalian nutrition because it provides vital nutrients for growth and development. Milk composition, which is influenced by genetic and environmental factors, supports lactation, a complex process crucial for milk production and quality. Recent research has focused on noncoding RNAs, particularly microRNAs (miRNAs), which are present in body fluids and regulate gene expression post-transcriptionally. This study comprehensively characterizes miRNAs in milk of four livestock species, namely Bubalus bubalis, Capra hircus, Equus asinus, and Ovis aries and identifies potential target genes. RESULTS: High-throughput sequencing of milk RNA resulted in distinct read counts across species: B. bubalis (8,790,441 reads), C. hircus (12,976,275 reads), E. asinus (9,385,067 reads), and O. aries (7,295,297 reads). E. asinus had the highest RNA mapping rate (94.6%) and O. aries the lowest (84.8%). A substantially greater proportion of miRNAs over other small RNAs was observed for the donkey milk sample (7.74%) compared to buffalo (0.87%), goat (1.57%), and sheep (1.12%). Shared miRNAs, which included miR-200a, miR-200b, miR-200c, and miR-23a among others, showed varying expression levels across species, confirmed by qPCR analysis. Functional annotation of predicted miRNA target genes highlighted diverse roles, with an enrichment in functions linked to metabolism and immunity. Pathway analysis identified immune response pathways as significant, with several miRNAs targeting specific genes across species, suggesting their regulatory function in milk. CONCLUSIONS: Both conserved and species-specific miRNAs were detected in milk of the investigated species. The identified target genes of these miRNAs have important roles in neonatal development, adaptation, growth, and immune response. Furthermore, they influence milk and meat production traits in livestock.

The concentrations of immunoglobulins in bovine colostrum determined by the gold standard method are genetically correlated with their near-infrared prediction.

BACKGROUND: The quality of colostrum administered to calves is based on its concentration in immunoglobulins G (IgG, g/L). Immunoglobulins A (IgA) and M (IgM) are also present but at a lower level. The gold standard reference analysis for these traits, radial immunodiffusion, is time-consuming and expensive. In order to define breeding strategies that are aimed at improving colostrum quality in dairy cattle, a large amount of data is needed, and the use of indicator traits would be beneficial. In the study presented here, we explored the heritabilities of reference (radial immunodiffusion) and near infrared-predicted IgG, IgA, and IgM concentrations and estimated their genetic correlations. First, the colostrum of 765 Holstein cows from nine herds was sampled to perform a reference analysis and the near-infrared spectra (400-2500 nm) were stored. We used a calibration set (28% of the initial samples) that was representative of the herds and cow parity orders to develop prediction equations for IgG, IgA, and IgM concentrations. Finally, these traits were predicted in the validation set (72% of the initial samples) to estimate genetic parameters for the predictions. Genetic correlations between reference and predicted values of each trait were estimated through bivariate linear animal models. RESULTS: The three near-infrared-predicted immunoglobulin fractions were genetically correlated with their reference value. In particular, the reference and predicted IgG concentrations were strongly correlated at both the genetic (0.854 ± 0.314) and phenotypic level (0.767 ± 0.019). Weaker associations were observed for IgA and IgM concentrations, which were predicted with lower accuracy compared to IgG. Simulation analyses suggested that improving colostrum quality by selective breeding in Holstein cattle based on near-infrared predicted colostrum immunoglobulins concentrations is feasible. In addition, less than 10 mL of colostrum are needed for spectra acquisition and thus implementation of such analyses is possible in the near future. CONCLUSIONS: The concentrations of colostrum immunoglobulins can be predicted from near-infrared spectra and the genetic correlation between the reference and the predicted traits is positive and favourable, in spite of the large standard errors of the estimates. Near-infrared spectroscopy can be exploited in selective breeding of dairy cattle to improve colostral immunoglobulins concentration.

Genetic and Non-Genetic Variation of Milk Total Antioxidant Activity Predicted from Mid-Infrared Spectra in Holstein Cows.

Food antioxidants enhance products shelf life and stability during technological treatments through the maintenance of their physical and chemical properties. Moreover, they are endowed with several positive effects on human health, including cell membranes preservation, enzyme functionality, and DNA integrity. Milk has been described in relation to a wide array of fat soluble and water-soluble antioxidant compounds, in particular vitamin A, C, and E, lactoferrin and peptides derived from casein and whey proteins. The total antioxidant activity (TAA) of milk is a novel and scarcely explored trait, defined as the sum of antioxidant contributions of the aforementioned compounds. On this background, the aims of the present study were to investigate the variability of milk TAA on a large scale exploiting predictions obtained through mid-infrared (MIR) spectroscopy and to estimate genetic parameters of this trait in Holstein cows. Individual milk samples were collected between January 2011 and December 2018 during the routine milk recording procedure. Samples were analysed for gross composition through MIR spectroscopy and MIR spectra were stored. Milk TAA was then predicted (pTAA) from the stored milk MIR spectra (111,653 test-day records of 9519 cows in 344 herds) using the previously developed prediction model; considering the prediction accuracy, pTAA might be considered a proxy of the TAA determined through the reference method. Overall, pTAA averaged 7.16 mmoL/L of Trolox equivalents, showed a nadir around 40 days after calving and increased thereafter, following a linear trend up to the end of lactation. The lowest pTAA was observed in milk sampled from June to September. Milk pTAA was heritable (0.401 ± 0.015) and genetically associated to fat yield (0.366 ± 0.049), crude protein (CP) yield (0.238 ± 0.052), fat percentage (0.616 ± 0.022) and CP percentage (0.754 ± 0.015). The official selection index of Italian Holstein put the 49% of the emphasis on fat and protein yield and percentage; therefore, it derives that an indirect favourable selection for milk pTAA should be already in progress in Italian Holstein population.

Variation of Blood Metabolites of Brown Swiss, Holstein-Friesian, and Simmental Cows.

Serum metabolic profile is a common method to monitor health and nutritional status of dairy cows, but blood sampling and analysis are invasive, time-consuming, and expensive. Milk mid-infrared spectra have recently been used to develop prediction models for blood metabolites. The current study aimed to investigate factors affecting blood ß-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), and urea nitrogen (BUN) predicted from a large milk mid-infrared spectra database. Data consisted of the first test-day record of early-lactation cows in multi-breed herds. Holstein-Friesian cows had the greatest concentration of blood BHB and NEFA, followed by Simmental and Brown Swiss. The greatest and the lowest concentrations of BUN were detected for Brown Swiss and Holstein-Friesian, respectively. The greatest BHB concentration was observed in the first two weeks of lactation for Brown Swiss and Holstein-Friesian. Across the first month of lactation, NEFA decreased and BUN increased for all considered breeds. The greatest concentrations of blood BHB and NEFA were recorded in spring and early summer, whereas BUN peaked in December. Environmental effects identified in the present study can be included as adjusting factors in within-breed estimation of genetic parameters for major blood metabolites.

Development of Infrared Prediction Models for Diffusible and Micellar Minerals in Bovine Milk.

Milk and dairy products are major sources of minerals in human diet. Minerals influence milk technological properties; in particular, micellar and diffusible minerals differentially influence rennet clotting time, curd firmness and curd formation rate. The aim of the present study was to investigate the ability of mid-infrared spectroscopy to predict the content of micellar and diffusible mineral fractions in bovine milk. Spectra of reference milk samples (n = 93) were collected using Milkoscan™ 7 (Foss Electric A/S, Hillerød, Denmark) and total, diffusible and micellar content of minerals were quantified using inductively coupled plasma optical emission spectrometry. Backward interval partial least squares algorithm was applied to exclude uninformative spectral regions and build prediction models for total, diffusible and micellar minerals content. Results showed that backward interval partial least squares analysis improved the predictive ability of the models for the studied traits compared with traditional partial least squares approach. Overall, the predictive ability of mid-infrared prediction models was moderate to low, with a ratio of performance to deviation in cross-validation that ranged from 1.15 for micellar K to 2.73 for total P.

Variation of Detailed Protein Composition of Cow Milk Predicted from a Large Database of Mid-Infrared Spectra.

This study aimed to investigate factors affecting protein fractions, namely α-casein (α-CN), ß-casein (ß-CN), κ-casein (κ-CN), ß-lactoglobulin (ß-LG) and α-lactalbumin (α-LA) predicted from milk infrared spectra in milk of dairy and dual-purpose cattle breeds. The dataset comprised 735,328 observations from 49,049 cows in 1782 herds. Results highlighted significant differences of protein fractions in milk of the studied breeds. Significant variations of protein fractions were found also through parities and lactation, with the latter thoroughly influencing protein fractions percentage. Interesting correlations (r) were estimated between ß-CN, κ-CN and ß-LG, expressed as percentage of crude protein, and milk urea nitrogen (r = 0.31, -0.20 and -0.26, respectively) and between α-LA and fat percentage (r = 0.41). The present study paves the way for future studies on the associations between protein fractions and milk technological properties, and for the estimation of genetic parameters of predicted protein composition.

Development of Fourier-transformed mid-infrared spectroscopy prediction models for major constituents of fractions of delactosated, defatted milk obtained through ultra- and nanofiltration.

Milk filtration procedures are gaining relevance in the dairy industry because milk ultra- and nanofiltrates are used to increase milk processing efficiency, and as additives for products with improved nutraceutical properties. This study aimed to develop Fourier-transformed mid-infrared spectroscopy calibrations for ultra- and nanopermeate and retentate fractions of defatted and delactosated milk. A total of 154 samples from different milk fractions were collected and analyzed using reference methods to determine protein, solids-not-fat, glucose, and galactose content. The obtained values were matched with their respective Fourier-transformed mid-infrared spectroscopy spectra to develop new prediction models. Calibrations for each trait were built following 3 different approaches to get the best prediction models: (1) using the entire data set, (2) using 3 subsets based on component concentrations (level approach), and (3) using hierarchical clusters calculated with pairwise Mahalanobis distance among spectra (cluster approach). Calibrations were developed using partial least squares regression, after removing low signal-to-noise ratio wavelengths, and validated through a leave-one-out cross-validation procedure. In addition, the accuracy of the predicted values within each fraction was checked for each approach. Dividing the data set into subsets improved prediction models for each trait and for the samples in each milk fraction. Without considering milk fraction, the best improvement was observed for glucose and galactose. Glucose ratio performance deviation in cross-validation (RPD) increased from 7.42 to 11.31 and 11.06, for cluster and level approaches, respectively, whereas galactose RPD increased from 8.86 to 11.69 and 11.27 for cluster and level approaches, respectively. Considering milk fractions, the best improvement was observed for protein content, where RPD ranged from 0.08 to 6.06 for the whole data set calibration, whereas it ranged from 0.43 to 40.34 for the subset calibration approaches. Cluster and level approaches to build calibration models were comparable for samples from different fractions, suggesting that the 2 subsetting protocols should be both investigated to get the best prediction performances.

Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.

A molecular dynamics strategy for CSαß peptides disulfide-assisted model refinement.

Many cysteine-stabilized antimicrobial peptides from a variety of living organisms could be good candidates for the development of anti-infective agents. In the absence of experimentally obtained structural data, peptide modeling is an essential tool for understanding structure-activity relationships and for optimizing the bioactive moieties. Focusing on cysteine-rich peptide structures, we reproduced the case of structure predictions in the so-called midnight zone. We developed our protocol on a training set derived by clustering the available cysteine-stabilized αß (CSαß) structures in nine different representative families and tested it on peptides randomly selected from each family. Starting from draft models, we tested a structure-based disulfide predictor and we used cysteine distances as constraints during molecular dynamics. Finally, we proposed an analysis for final structure selection. Accordingly, we obtained a mean root mean square deviation improvement of 21% for the test set. Our findings demonstrate that it is possible to predict the network of disulfide bridges in cysteine-stabilized peptides and to use this result to improve the accuracy of structural predictions. Finally, we applied the methods to predict the structure of royalisin, a cysteine-rich peptide with unknown structure.

Structural and Antimicrobial Features of Peptides Related to Myticin C, a Special Defense Molecule from the Mediterranean Mussel Mytilus galloprovincialis.

Mussels (Mytilus spp.) have a large repertoire of cysteine-stabilized α,ß peptides, and myticin C (MytC) was identified in some hundreds of transcript variants after in vivo immunostimulation. Using a sequence expressed in Italian mussels, we computed the MytC structure and synthesized the mature MytC and related peptide fragments (some of them also prepared in oxidized form) to accurately assess their antibacterial and antifungal activity. Only when tested at pH 5 was the reduced MytC as well as reduced and oxidized fragments including structural ß-elements able to inhibit Gram-positive and -negative bacteria (MIC ranges of 4-32 and 8-32 µM, respectively). Such fragments caused selective Escherichia coli killing (MBC of 8-32 µM) but scarcely inhibited two fungal strains. In detail, the antimicrobial ß-hairpin MytC[19-40]SOX caused membrane-disrupting effects in E. coli despite its partially ordered conformation in membrane-mimetic environments. In perspective, MytC-derived peptides could be employed to protect acidic mucosal tissues, in cosmetic and food products, and, possibly, as adjuvants in aquaculture.