Spectral Profiling (Fourier Transform Infrared Spectroscopy) and Machine Learning for the Recognition of Milk from Different Bovine Breeds.

The Podolica cattle breed is widespread in southern Italy, and its productivity is characterized by low yields and an extraordinary quality of milk and meats. Most of the milk produced is transformed into "Caciocavallo Podolico" cheese, which is made with 100% Podolica milk. Fourier Transform Infrared Spectroscopy (FTIR) is the technique that, in this research work, was applied together with machine learning to discriminate 100% Podolica milk from contamination of other Calabrian cattle breeds. The analysis on the test set produced a misclassification percentage of 6.7%. Among the 15 non-Podolica samples in the test set, 2 were misclassified and recognized as Podolica milk even though the milk was from other species. The correct classification rate improved to 100% when the same method was applied to the recognition of Podolica and Pezzata Rossa milk produced by the same farm. Furthermore, this technique was tested for the recognition of Podolica milk mixed with milk from other bovine species. The multivariate model and the respective confusion matrices obtained showed that all the 14 Podolica samples (test set) mixed with 40% non-Podolica milk were correctly classified. In addition, Pezzata Rossa milk produced by the same farm was detected as a contaminant in Podolica milk from the same farm down to concentrations as little as 5% with a 100% correct classification rate in the test set. The method described yielded higher accuracy values when applied to the discrimination of milks from different breeds belonging to the same farm. One of the reasons for this phenomenon could be linked to the elimination of the environmental variable. However, the results obtained in this work demonstrate the possibility of using FTIR to discriminate between milks from different breeds.

The Bee Gut Microbiota: Bridging Infective Agents Potential in the One Health Context.

The bee gut microbiota plays an important role in the services the bees pay to the environment, humans and animals. Alongside, gut-associated microorganisms are vehiculated between apparently remote habitats, promoting microbial heterogeneity of the visited microcosms and the transfer of the microbial genetic elements. To date, no metaproteomics studies dealing with the functional bee microbiota are available. Here, we employ a metaproteomics approach to explore a fraction of the bacterial, fungal, and unicellular parasites inhabiting the bee gut. The bacterial community portrays a dynamic composition, accounting for specimens of human and animal concern. Their functional features highlight the vehiculation of virulence and antimicrobial resistance traits. The fungal and unicellular parasite fractions include environment- and animal-related specimens, whose metabolic activities support the spatial spreading of functional features. Host proteome depicts the major bee physiological activities, supporting the metaproteomics strategy for the simultaneous study of multiple microbial specimens and their host-crosstalks. Altogether, the present study provides a better definition of the structure and function of the bee gut microbiota, highlighting its impact in a variety of strategies aimed at improving/overcoming several current hot topic issues such as antimicrobial resistance, environmental pollution and the promotion of environmental health.

Recent Advances in the Determination of Milk Adulterants and Contaminants by Mid-Infrared Spectroscopy.

The presence of chemical contaminants, toxins, or veterinary drugs in milk, as well as the adulteration of milk from different species, has driven the development of new tools to ensure safety and quality. Several analytical procedures have been proposed for the rapid screening of hazardous substances or the selective confirmation of the authenticity of milk. Mid-infrared spectroscopy and Fourier-transform infrared have been two of the most relevant technologies conventionally employed in the dairy industry. These fingerprint methodologies can be very powerful in determining the trait of raw material without knowing the identity of each constituent, and several aspects suggest their potential as a screening method to detect adulteration. This paper reviews the latest advances in applying mid-infrared spectroscopy for the detection and quantification of adulterants, milk dilution, the presence of pathogenic bacteria, veterinary drugs, and hazardous substances in milk.

The Effect of Feeding a Total Mixed Ration with an ad libitum or Restricted Pelleted Starter on Growth Performance, Rumination Behavior, Blood Metabolites, and Rumen Fermentation in Weaning Holstein Dairy Calves.

The aim of the current study was to evaluate the effect of the starter restriction and of the ad libitum TMR (total mixed ration) inclusion on intake, growth performance, rumination time (RT), and health condition of Holstein dairy calves during weaning. We randomly assigned thirty female Holstein calves (with an average weight of 38.5 ± 1.96 kg at birth) to one of three treatments. From 21 days of age, the calves were fed one of three treatments as follows: a control diet (CTR) with an ad libitum calf starter but without TMR; Treatment 1 diet (TRT1) with both an ad libitum calf starter and ad libitum TMR; Treatment 2 diet (TRT2) with ad libitum TMR and a restricted amount of a calf starter (50% of the intake recorder in the control group day by day). Calves in the TRT2 group, between 56 and 63 days of age, had a lower body weight (80.1; 79.5; 75.6 kg for the CTR, TRT1, and TRT2 groups, respectively) compared with CTR and TRT1 calves. This outcome is ascribed to the average daily gain (0.759; 0.913; 0.508 kg/day for the CTR, TRT1, and TRT2 groups, respectively), resulting also in TRT2 being lower than CTR or TRT1 calves. The inclusion of ad libitum TMR increased the rumination time, especially after weaning (15.28 min/h, 18.38 min/h, and 18.95 min/h for the CTR, TRT1, and TRT2 groups, respectively). Concerning the rumen metabolism and inflammometabolic response, overall, no differences were observed between the three dietary treatments. In conclusion, the results indicated that a TMR could partially replace a calf starter in weaning dairy calves, since neither growth performance nor health status were impaired. In addition, providing TMR (with or without concentrate restriction) led to a better rumen development and likely a better rumen fermentation efficiency in post-weaning.

The Functional Characteristics of Goat Cheese Microbiota from a One-Health Perspective.

Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese.

Mid-infrared (MIR) spectroscopy for the detection of cow's milk in buffalo milk.

In Italy, buffalo mozzarella is a largely sold and consumed dairy product. The fraudulent adulteration of buffalo milk with cheaper and more available milk of other species is very frequent. In the present study, Fourier transform infrared spectroscopy (FTIR), in combination with multivariate analysis by partial least square (PLS) regression, was applied to quantitatively detect the adulteration of buffalo milk with cow milk by using a fully automatic equipment dedicated to the routine analysis of the milk composition. To enhance the heterogeneity, cow and buffalo bulk milk was collected for a period of over three years from different dairy farms. A total of 119 samples were used for the analysis to generate 17 different concentrations of buffalo-cow milk mixtures. This procedure was used to enhance variability and to properly randomize the trials. The obtained calibration model showed an R 2 ≥ 0.99 (R 2cal. = 0.99861; root mean square error of cross-validation [RMSEC] = 2.04; R 2val. = 0.99803; root mean square error of prediction [RMSEP] = 2.84; root mean square error of cross-validation [RMSECV] = 2.44) suggesting that this method could be successfully applied in the routine analysis of buffalo milk composition, providing rapid screening for possible adulteration with cow's milk at no additional cost.

The Quality of Five Natural, Historical Italian Cheeses Produced in Different Months: Gross Composition, Fat-Soluble Vitamins, Fatty Acids, Total Phenols, Antioxidant Capacity, and Health Index.

Five natural historic cheeses of Southern Italy were investigated-Caciocavallo Palermitano (CP), Casizolu del Montiferru (CdM), Vastedda della Valle del Belìce (VVB), Pecorino Siciliano (PS), and Caprino Nicastrese (CN)-which are produced with raw milk and with traditional techniques and tools, from autochthonous breeds reared under an extensive system. The effects of the month of production on gross composition, MUFA, PUFA, PUFA-ω6, PUFA-ω3, α-tocopherol, retinol, cholesterol, TPC, TEAC, and GHIC were evaluated. In CP, CLA, TPC, and GHIC were higher in April than in February. CdM showed higher values in terms of fat, saturated fatty acids, PUFA-ω3, α-tocopherol, TEAC, and GHIC in May than in February and September, while low values in terms of protein, moisture, and CLA were found. In VVB, MUFA, PUFA-ω6, and α-tocopherol increased in June compared with April; conversely, protein, FRAP, and TEAC were higher in April. In PS, protein, CLA, PUFA, PUFA-ω3, α-tocopherol, and GHIC increased in May compared with January; on the contrary, moisture, NaCl, and TEAC showed high values in January. CN showed higher values in terms of PUFA, PUFA-ω6, PUFA-ω3, TPC, TEAC, and GHIC in April and June compared with January. It is shown that each cheese is unique and closely linked to the production area. Cheeses produced in the spring months showed a high nutritional quality due to the greatest presence of healthy compounds originating from an extensive feeding system.

Fungal Contamination and Aflatoxin B1 Detected in Hay for Dairy Cows in South Italy.

The growth of filamentous fungi on fodder is recognized as responsible for fungal deterioration and mycotoxin contamination of the plant mass leads to economic losses in the dairy cow production system. Mycotoxin contamination has significant implications for human and animal health and is one of the major concerns in the food and feed chain. This research provides an insight into the variety of viable molds (i.e., filamentous microfungi) that can be isolated from hay produced in South Italy and destined to dairy cows. On different lots of hay (n = 55) collected from 20 dairy farms, a total of 33 different fungal species were identified. The most representative was Cladosporium cladosporioides (n = 46, 84%) followed by Alternaria alternata (n = 25, 45%), and Rhizopus stolonifer (n = 24, 44%). The species most closely related to aflatoxin (AF) contamination, Aspergillus flavus, was often isolated (n = 11, 20%). Regarding AF detection, all the hay samples were found to be scarcely contaminated by AFB1 and showed values from 0.0020 to 0.0077 mg/kg, below the limits established by European Union (EU legislation) (0.02 mg/kg). None of the samples were positive for Aspergillia and tested for AFB1 showed results exceeding established limits. Additionally, hay with moisture between 15.0 and 19.2% or crude ash on dry matter content ranging from 14.0 to 15.5% reported an increased presence of AFB1 (p < 0.05) compared to the other samples. All the analyzed hay samples, besides the presence of molds, can be considered safe for the presence of AFB1. Prevention of mold spoilage is mandatory to reduce the exposure of humans and animals to mycotoxins.

Suitability of Protein Content Measured by MilkoScan FT-Plus Milk Analyzer to Evaluate Bovine and Ovine Colostrum Quality.

The objective of this study was to evaluate MilkoScan FT-plus for the estimation of the immunoglobulin G (IgG) content in bovine and ovine colostrum. Between April and May 2016, a total of 94 colostrum samples (54 from Simmental dairy cows and 39 from Sarda ewes) were collected within 6 h (T0) and after 24 h (T24) from parturition. Colostrum samples were subjected to the radial immunodiffusion (RID) assay for the quantification of IgG and to MilkoScan FT-plus for the estimation of protein content (TP, %), which was then used as an indirect method for the evaluation of colostrum quality. To compare the two methods, correlation and regression analysis of IgG quantification by RID and protein (%) content estimation by MilkoScan FT-plus data was performed using Procedure CORR and Procedure REG of SAS, respectively (version 9.3, SAS Institute Inc., Cary, NC, USA). Thresholds for the classification of good colostrum quality (as determined by RID assay, the gold standard method) were set at 50 g of IgG/L in cows and 20 g of IgG/L in ewes. The concentration of IgG in bovine colostrum assayed by RID showed a variation ranging from 41.45 to 199.97 g/L with an average of 99.85 ± 40.84 g/L at T0, and from 2.83 to 75.93 g/L with an average of 19.76 ± 19.01 g/L at T24. Regarding ovine colostrum, the concentration of IgG assayed by RID ranged from 34.45 to 156.32 g/L with an average value of 77.82 ± 37.58 g/L at T0, and from 5.6 to 69.74 g/L with an average of 27.90 ± 19.81 g/L at T24. Colostrum TP ranged from 3.70 to 23.96% for bovine colostrum and 6.32 to 22.88% for ovine colostrum using MilkoScan FT-plus. MilkoScan FT-plus and RID data were highly and significantly correlated (r = 0.91 for bovine and r = 0.94 for ovine colostrum), and regression analysis showed a strong relationship between IgG concentration provided by RID assay and TP provided by MilkoScan FT-plus (R2 = 0.84 and 0.88 for bovine and ovine, respectively). Optimal cut-off points for the greatest accuracy of TP (%) determined by MilkoScan FT-plus were 12.8% in cows [with 88.9% sensitivity (Se) and 100% specificity (Sp)] and 9% in ewes (with 96.7% Se and 100% Sp). In conclusion, these outcomes indicate that MilkoScan FT-plus as an indirect method may be a reliable tool for the estimation of the total IgG concentration and quality in bovine and ovine colostrum. Moreover, the cut-off levels of 12.8% for bovine and 9% for ovine of TP, seem sufficient to ensure that all poor-quality colostrum can be classified as such, with only a low proportion of good-quality colostrum being misclassified as poor-colostrum, thereby increasing the probability of delivering good-quality colostrum to new-born calves and lambs.

Effects of ecotrofin™ on milk yield, milk quality and serum biochemistry in lactating goats.

A nutritional supplement (Ecotrofin™, by Vetoquinol Italia S.r.l) recommended in ruminants feeding to strengthen the physiological condition and improve digestive performance was tested in 20 pluriparae grazing goats divided in two groups (control and treated) to assess its possible effects on milk yield and quality and to assess eventual adverse effects. Animals from both groups also received 400 g/day of corn meal, and the treated group was supplemented with 20 g/head/day of the nutritional supplement. At the doses suggested by the manufacturer, despite a transient increase after 30 days of supplementation, Ecotrofin™ did not show significant effects on milk yield and, although some changes were found in the fatty acids profile, no significant improvement of MUFA and PUFA, as well as of omega-6:omega-3 ratio and CLA content were seen. Therefore, in our experimental conditions the supplementation of diet with Ecotrofin™ did not appear useful to improve goat's performance. A significant effect on kidney health markers (27 vs. 22.5 for urea and 0.83 vs. 0.76 for creatinine, p < 0.05) suggested a beneficial effect on renal function but, since levels fell in the normal ranges in both groups, such hypothesis would need further studies to be addressed.

Pegbovigrastim Treatment around Parturition Enhances Postpartum Immune Response Gene Network Expression of whole Blood Leukocytes in Holstein and Simmental Cows.

Pegbovigrastim is a commercial long-acting analog of bovine granulocyte colony-stimulating factor (rbG-CSF) that promotes the increased count and functionality of polymorphonuclear cells in dairy cows around the time of parturition. We hypothesized that pegbovigrastim administered to periparturient cows at approximately seven days before parturition and within 24 hours after calving could affect the profiles of gene networks involved in leukocyte function. Blood was collected on Day 3 after calving from treated groups (pegbovigrastim (PEG); 13 Simmental (seven multiparous and six primiparous) and 13 Holstein (seven multiparous and six primiparous) cows) that received pegbovigrastim (Imrestor; Elanco Animal Health) and controls (CTR; 13 Simmental (seven multiparous and six primiparous) and 13 Holstein (six multiparous and seven primiparous) cows) that received saline solution. Blood from all cows was sampled from the jugular vein in a PAXgene Blood RNA System tube (Preanalytix, Hombrechtikon, Switzerland) for RNA extraction. The RT-qPCR analysis was performed to investigate a panel of 34 genes of interest, representing recognition, immune mediation, migration, cell adhesion, antimicrobial strategies, inflammatory cascade, oxidative pattern, and leukotrienes in whole blood leukocytes. Normalized data were subjected to the MIXED model of SAS (ver. 9.4) with treatment, breed, parity, and their interaction as fixed effects. Compared with CTR, whole blood leukocytes of PEG cows had higher expression of genes involved in recognition and immune modulation (CD14, CD16, MYD88, TLR2, and TLR4), cell adhesion (ITGB2, ITGAL, TLN1, SELL, SELPLG, and CD44), antimicrobial activity (MMP9, LTF, and LCN2), and inflammatory cascade (CASP1, TNFRSF1A, IL1B, IL1R, IL18, IRAK1, NLRP3, and S100A8). This suggested an improvement of migration, adhesion, and antimicrobial ability and an enhanced inflammatory response, which in turn could trigger immune cell activation and enhance function. Expression of SOD2 and ALOX5 was also greater in the PEG group. In contrast, compared with CTR cows, PEG led to lower expression of RPL13A, ALOX15, IL8, and TNF. Overall, leukocytes from Simmental compared with Holstein cows had greater expression of IDO1, RPL13A, ALOX5, CD44, CX3CR1, ITGB2, and TNFA, whereas expression of CD16 and TLR2 was lower. Overall, compared with multiparous cows, primiparous cows had higher expression of IL1B, IL18, MYD88, SELL, and TLR2 and lower expression of MMP9. Simmental cows seemed more sensitive to induction of the immune system after calving, as revealed by the greater abundance of genes involved in immune system adaptation, regardless of pegbovigrastim treatment. Primiparous cows undergoing a new stress condition with respect to older cows were characterized by leukocytes with a higher inflammatory response. In conclusion, pegbovigrastim led to higher expression levels of most genes involved in the processes investigated, suggesting a thorough activation of the immune machinery during the critical post-partum period.

Milk microbiota: Characterization methods and role in cheese production.

Milk is a complex body fluid aimed at addressing the nutritional and defensive needs of the mammal's newborns. Harbored microbiota plays a pivotal role throughout the cheesemaking process and contributes to the development of flavor and texture typical of different type of cheeses. Understanding the dairy microbiota dynamics is of paramount importance for controlling the qualitative, sensorial and biosafety features of the dairy products. Although many studies investigated the contribution of single or few microorganisms, still there is some information lacking about microbial communities. The widespread of the omics platforms and bioinformatic tools enable the investigation of the cheese-associated microbial community in both phylogenetical and functional terms, highlighting the effects of the diverse cheesemaking variables. In this review, the most relevant literature is revised to provide an introduction of the milk- and cheese-associated microbiota, along with their structural and functional dynamics in relation to the diverse cheesemaking technologies and influencing variables. Also, we focus our attention on the latest omics technologies adopted in dairy microbiota investigation. Discussion on the key-steps and major drawbacks of each omics discipline is provided along with a collection of results from the latest research studies performed to unravel the fascinating world of the dairy-associated microbiota. SIGNIFICANCE: Understanding the milk- and cheese- associated microbial community is nowadays considered a key factor in the dairy industry, since it allows a comprehensive knowledge on how all phases of the cheesemaking process impact the harbored microflora; thus, predict the consequences in the finished products in terms of texture, organoleptic characteristics, palatability and biosafety. This review, collect the pioneering and milestones works so far performed in the field of dairy microbiota, and provide the basic guidance to whom approaching the cheese microbiota investigation by means of the latest omics technologies. Also, the review emphasizes the benefits and drawbacks of the omics disciplines, and underline how the integration of diverse omics sciences enhance a comprehensive depiction of the cheese microbiota. In turn, a better consciousness of the dairy microbiota might results in the application of improved starter cultures, cheesemaking practices and technologies; supporting a bio-safe and standardized production of cheese, with a strong economic benefit for both large-scale industries and local traditional dairy farms.

Unraveling the Adipose Tissue Proteome of Transition Cows through Severe Negative Energy Balance.

Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points (one month before calving, one and sixteen weeks after calving) that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins (annexin A2, actin-related protein 10, glyceraldehyde-3-phosphate dehydrogenase, and fatty acid-binding protein) involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum.