Dairy

Effective colostrum management is essential to ensure adequate transfer of passive immunity (TPI) and reduce neonatal morbidity in calves. This study evaluated the impact of pasteurization on colostrum microbiological and immunological quality and its association with TPI and calf health on a dairy farm in Italy. A total of 79 colostrum samples were included: 39 pasteurized (PC) and 40 non-pasteurized (NPC). Immunoglobulin concentration was estimated by Brix refractometry, while bacterial contamination was assessed by total bacterial count (TBC) and total coliform count (TCC). In a second phase of the study, 80 newborn calves received either pasteurized (C-PC, n = 40) or non-pasteurized colostrum (C-NPC, n = 40). TPI was evaluated at 48 h and calves were clinically monitored for 21 days. Overall, colostrum showed not only high Brix values but also elevated bacterial contamination. Pasteurization significantly reduced the microbial load in colostrum (TBC, 5.7 vs. 2.9 log CFU/mL; TCC 4.6 vs. 1.5 log CFU/mL) (p < 0.0001). Brix values differed significantly between PC and NPC colostrum (p < 0.001), although the magnitude of the difference was minimal (27.8% in NPC vs. 27.6% in PC), suggesting limited biological relevance. Calves receiving pasteurized colostrum showed significantly lower odds of developing neonatal diarrhea than calves receiving unpasteurized colostrum (C-PC: OR 0.1, 95% CI 0.0–0.3; C-NPC: OR 9.9, 95% CI 3.4–32.6). These findings indicate that colostrum pasteurization reduces contamination effectively even in colostrum with high bacterial loads without substantial impact on its Brix values and was associated with improved calf diarrhea outcomes in the investigated herd. Full article

Chemical contamination of milk represents a relevant food safety concern, particularly in countries where systematic monitoring programs are still limited. In Algeria, information on human exposure to aflatoxin M₁ (AFM₁) and per- and polyfluoroalkyl substance (PFAS) contamination remains fragmented or lacking. This study assessed the occurrence of AFM₁ and 17 PFAS in cow and goat milk and explored farm-level management practices potentially influencing contamination. Bulk-tank milk samples were collected from 26 dairy cattle and 11 dairy goat farms. Farm characteristics, including feeding strategies, water sources, grazing practices, and herd size, were recorded using structured questionnaires to characterize farm-level practices and explore their potential association with contamination patterns. AFM₁ was detected in all samples. In cow milk, 96.1% exceeded the EU limit (0.05 µg/kg), with concentrations ranging from 0.048 to 0.410 µg/kg (mean: 0.102 µg/kg), although none exceeded the Codex Alimentarius limit (0.50 µg/kg). Goat milk showed a lower prevalence of samples above the EU limit (36.4%), but higher concentrations were observed (range 0.030–0.578 µg/kg; mean 0.193 µg/kg), with two samples (18.2%) exceeding the Codex limit. PFAS contamination was generally low: 73.0% of cow and 64.0% of goat samples were below detection limits, with only PFBS (cow milk) and PFDA (goat milk) quantified above LOQ. Because of the limited number of farms and the single sampling period, no robust associations could be established between farm-level variables and contaminant concentrations. This preliminary study provides an exploratory overview of AFM₁ and PFAS occurrence in bovine and goat bulk milk from the sampled Algerian farms. These findings reveal widespread AFM₁ contamination within the investigated farms, consistent with possible feed-borne AFB₁ exposure, whereas PFAS occurrence appeared sporadic and generally low. Full article

Postpartum dairy cows fed more rapidly fermentable starch sources have depressed dry matter intake (DMI), compounding the risk of negative energy balance and hepatic lipid accumulation. Rumen-protected choline (RPC) is supplemented to periparturient dairy cows to facilitate hepatic lipid export. Our objective was to evaluate the interaction of dietary starch fermentability (DF) and RPC supplementation on postpartum DMI and performance. Prepartum supplementation of a low dose of RPC (no RPC [C−] vs. RPC [C+; 30 g/d]) began 21 d before the expected calving date for Holstein cows with at least one parity. Postpartum, cows were assigned to 1 of 4 postpartum treatments for 21 d with a 2 × 2 factorial arrangement of starch fermentability rate (low [dry-rolled corn; LFERM] vs. high [dry-rolled wheat; HFERM]) and RPC (C− vs. C+). Prepartum, C+ decreased DMI by 2.3 kg compared with C−, but there was no evidence of treatment effect on DMI postpartum. Time and DF interacted on milk yield, with HFERM increasing milk yield after d 3 compared with LFERM. Compared with LFERM, HFERM decreased milk fat content but not fat yield. For blood metabolites, C+ decreased plasma beta-hydroxybutyrate by 0.3 mmol/L and tended to increase glucose concentration compared to C−. In conclusion, supplementation with RPC at a low rate of inclusion reduced DMI in prepartum cows and decreased postpartum plasma BHB concentrations. Further work is required to elucidate potential mechanisms of action for RPC-mediated reductions in DMI. Full article

Heat stress (HS) is a major issue affecting productivity and physiological responses in dairy cattle. Advances in gene function research have made it possible to identify favorable genotypes for HS tolerance. This study evaluates the combined effects of temperature–humidity index (THI) and genomic load of favorable SNPs in the genes GRM8, SMAD3, and TLR4 on daily milk yield (DMY), rectal temperature (RT), and gene expression in Holstein cows (n = 160). Environmental conditions ranged from thermoneutral to severe HS, allowing the assessment of genotype by environment interactions. A subset of cows (n = 40) was selected based on genomic load to evaluate gene expression under opposite thermal conditions. Results showed that increasing THI was associated with reduced DMY and increased RT, with more pronounced effects in cows carrying fewer favorable genotypes. Slope analyses revealed that cows with higher genomic load exhibited a moderate decline in DMY and lower increase in RT, indicating enhanced resilience to HS. Significant THI × genomic load interactions were detected above a threshold of 79 units (p < 0.05). Gene expression analyses supported these findings, showing differential expressions of GRM8, SMAD3, and TLR4 under HS conditions. The functional and predictive value of these genes as markers of thermotolerance could help producers by supporting their application in genetic selection programs, thus providing another tool to combat HS and improve resilience and productivity in dairy systems. Full article

This study investigated the impact of moderate teat-end hyperkeratosis on milk somatic cell count (SCC) and bacterial count (BC) in Holstein cows. Comparing teat-end scores (TS) 2 and 3, we found that TS3 quarters exhibited significantly higher SCC (p = 0.017) despite having shorter days in milk than TS2 quarters. While BC in foremilk (BC1) remained stable, post-milking samples (BC2) decreased during sequential sampling; however, a significant Group × Time interaction (p = 0.020) revealed a less pronounced bacterial reduction in TS3. These findings tentatively suggest that moderate hyperkeratosis (TS3) may be associated with subclinical mammary irritation and altered bacterial clearance dynamics, independent of the absolute bacterial load. Managing teat-end morphology may help optimize milk quality, even when bacterial contamination is minimal. Full article

The dairy industry is increasingly seeking natural alternatives to synthetic additives to meet the growing demand for clean-label and functional foods. Bee-derived products (BDPs), including propolis, honey, bee pollen, bee bread, and royal jelly, represent a promising class of bioactive ingredients due to their antimicrobial, antioxidant, and immunomodulatory properties. This review critically examines their integration across the dairy value chain, adopting a farm-to-product perspective. At the farm level, BDPs can modulate rumen fermentation, influence microbial populations, and contribute to improved feed efficiency and reduced enteric methane emissions. These effects may translate into modifications in milk composition and functional properties. At the processing and product levels, the incorporation of BDPs into dairy matrices such as yogurt, cheese, and fermented milk enables the development of functional foods enriched with bioactive compounds and supports probiotic viability in synbiotic systems. However, their application is associated with technological and sensory challenges, including variability in chemical composition, dose-dependent antimicrobial effects, and potential impacts on texture and flavour. By bridging animal and food science, this review highlights the multifunctional role of BDPs in enhancing sustainability, safety, and nutritional value in dairy systems, while identifying current limitations and future research directions for their effective industrial implementation. Full article

Breed and herd are major determinants of milk productivity and efficiency. This study evaluated 1508 lactating cows from three dairy (Holstein, Brown Swiss, Jersey) and three dual-purpose (Simmental, Rendena, Alpine Grey) breeds across 41 multi-breed mountain herds. Data on body size, production, and milk samples were collected. Daily milk yield, milk energy, and cheese yield were quantified, and herds were classified under high (>75 MJ/d) or low (<75 MJ/d) average daily milk energy production. Cheese-making traits were predicted from 508 individual model cheese-making trials. Nine productivity ratios and three efficiency indicators (energy efficiency, economic efficiency, and income over feed costs) were calculated. Data was analyzed using mixed models including herd productivity class, herd within class (random), breed, parity, and days in milk. Differences among breeds were smaller within herds than across herds and were more pronounced for quality and cheese-making traits. Dairy and dual-purpose breeds showed similar overall performance. Jerseys were the least productive in absolute terms but, when adjusted for body size, were as or more efficient than other dairy breeds. Holsteins had the highest milk yield, whereas Brown Swiss showed superior milk quality and cheese yield. Simmental outperformed local dual-purpose breeds in size and production but not in efficiency. Accurate assessment of energy and economic efficiency is essential for breeding and crossbreeding strategies. Full article

Mycotoxins are toxic fungal secondary metabolites that can contaminate the feed of dairy animals and can be transferred into milk and dairy products. This study aims to assess the occurrence of major mycotoxins in 190 samples of milk-based infant foods commercially available in São Paulo, Brazil, and characterize the associated risks. Aflatoxins (AFs) M₁ (AFM₁), B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂), fumonisins (FBs) B₁ (FB₁) and B₂ (FB₂), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), and T-2 toxin were analyzed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS). Ninety-eight samples (51.6%) were contaminated with at least one mycotoxin, while the co-occurrence of two to four mycotoxins was observed in 32 samples (16.8%). ZEN was the most prevalent mycotoxin (60 samples; 32%), followed by FBs (FB₁ and FB₂) (42 samples; 22%), OTA (11 samples; 5.8%), DON (8 samples; 4.2%), and T-2 toxin (2 samples; 1.1%). AFs were quantified in only two samples (1.1%), one milk-based product containing 0.630 μg/kg of AFM₁ and one flavored milk powder with AFB₁ at 5.850 μg/kg. The analyzed flavored milk powder samples showed the highest maximum concentrations of DON, FBs, and OTA (165.5, 93.79, and 86.77 μg/kg, respectively), while unflavored milk powder contained the highest concentration of ZEN (17.40 μg/kg). Infant formula provided the highest estimated daily intake (EDI) value of DON (1.226 μg/kg bw/day) for infants aged ≤one year. Hazard quotient (HQ) values > 1 revealed a potential health concern for DON in only one sample of infant formula intended for infants up to six months of age. Margin of exposure (MoE) values for AFs and OTA were <10,000, emphasizing the need for continuous monitoring due to their potential toxic effects. These findings emphasize the need for stringent preventive measures and regulatory compliance to prevent mycotoxin contamination in milk-based infant foods in Brazil. Full article

This study evaluated the effects of replacing alfalfa hay with sulla [Sulla coronaria (L.) B.H. Choi & H. Ohashi] hay or sulla haylage in pasture-based diets for lactating Sarda ewes on milk production, milk composition, and nutrient utilization efficiency. Seventy-two mid-lactation ewes were assigned to three dietary treatments for 56 days—alfalfa hay (AH), sulla hay (SH), or sulla haylage (SHL)—all combined with 3 h/day grazing and a fixed amount of supplemental concentrate. The diets were formulated to be theoretically isoenergetic and isoproteic. Pasture nutritive value was generally comparable among the groups throughout the experimental period. Milk yield did not differ among treatments; however, fat- and protein-corrected milk (FPCM) and milk fat and protein concentrations were higher in AH compared with the sulla-based diets. Actual nutrient intake differed among treatments, with nitrogen intake (NI) being greatest in AH, consistent with the higher milk urea concentration observed in this group. Estimated apparent energy utilization efficiency (FPCM/UFL intake) showed treatment-associated differences in exploratory analyses, with lower values observed in SH compared with AH and SHL. Similarly, exploratory estimates of apparent nitrogen utilization efficiency (Milk N/NI) were highest in SHL, intermediate in SH, and lowest in AH (p < 0.001). Condensed tannins were not detected in the conserved sulla forages under the analytical conditions adopted in this study. Therefore, the observed responses were unlikely to be directly associated with detectable condensed tannin activity. Overall, the results suggest that forage conservation method may influence milk composition and estimated apparent nutrient utilization indices, with sulla haylage showing higher exploratory estimates of apparent nitrogen utilization efficiency without negative effects on milk yield under the conditions of the present study. Full article

The intensification and continuous evolution of dairy sheep and goat farming have played an essential role in the development and implementation of milking equipment. The increasing demand for time-efficient milking procedures, reduced labour costs, sustained milk production, and optimal mammary health have driven the widespread adoption and optimisation of machine milking technologies. The objectives of this article are (i) the review of milking systems and relevant technological developments in milking equipment and (ii) the evaluation and description of their impact on udder health, as applied on dairy small ruminant farms. Milking systems used on farms depend on the available space and number of animals on the farms. Appropriate settings in milking systems are important for ensuring good milk quality; among them, vacuum level, pulsation rate and ratio are important characteristics that must be monitored regularly. Further, use of appropriate teatcups specific to the animal species to be milked is significant. An important aspect of proper maintenance of the milking system is the cleaning procedure after completion of milking. Points for consideration are quality and temperature of the water used for cleaning, use of detergents and disinfectants, and maintenance schedule and teatcup replacement. Some technological features that are part of milking systems include automatic vacuum shut off, electronic milk recording, electronic identification of animals, automatic flushing of milking clusters and automatic pre-stimulators. Farms will benefit from applying precision technologies, which will use data from tools related to animal genetic background, animal behavioural indicators, environmental conditions and disease-related functions for more holistic and cost-effective farm management. In this context, integration of sensor-based technologies in milking systems will be able to provide real-time information regarding quality of milk produced at individual and farm levels. Moreover, the introduction of automatic system flushing in-between animals during the milking procedure can contribute to breaking chains of potential bacterial transfer and reducing animal infections during milking. Overall, although machine milking has certainly contributed to improved efficiency, milk quality and labour conditions, flaws in system function may adversely affect mammary health. Full article

Automatic milking systems (AMSs) are increasingly adopted in dairy farms, and barn design, particularly regarding cow traffic design (CowTD), plays a key role in system performance. This study evaluated the association between different CowTDs and operational and production indicators of AMS Brazilian dairy farms. The data were obtained from 149 commercial dairy farms equipped with AMS (average of 1.6 AMS per farm) encompassing approximately 14,642 lactating cows recorded between June and December 2025. Cow traffic designs were classified as free or smart-gated systems, including milk-first (MF) and feed-first (FF) configurations. Mixed models were used to evaluate the effects of regions, housing system, and CowTD on the number of lactating cows per AMS (NCowsAMS), milking frequency (MFreq), milk yield per milking (MYMilking), daily milk yield per cow (MYcow), daily milkings per AMS (MilkingsAMS), and daily milk yield per AMS (MYAMS). On average, farms milked 58 cows per AMS with a mean MFreq of 2.69 milkings/cow per day and produced 2227 kg of milk per AMS per day. Smart-gated CowTD supported a greater number of cows per AMS than free CowTD systems (FF = 57 and MF = 58 vs. Free = 53 cows/AMS). Although free CowTD increased MFreq to approximately three milkings/cow/day, this advantage did not translate into greater or equivalent MYAMS, despite MF and free CowTD exhibiting similar MYcow (37.0 and 37.2 kg/day, respectively). Even though free CowTD achieved the highest MilkingsAMS (Free = 156 vs. MF = 151 and FF = 143 milkings/day), it milked fewer cows per robot, resulting in lower MYAMS. Consequently, FF and MF systems produced 86 and 180 kg/day more MYAMS, respectively, than free CowTD. These results suggest that AMS performance is primarily driven by the NCowsAMS rather than MFreq alone. Under Brazilian commercial conditions, smart-gated CowTD systems appear to be more efficient, as evidenced by higher MYAMS, while allowing higher stocking densities, potentially without increasing labor requirements. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects 25–30% of adults globally. Dietary sugar reduction is one of the key therapeutic targets, but elimination of sugar-sweetened foods may compromise adherence to calorie-restricted diets. Brazzein, a natural sweet protein that is 500–2000 times sweeter than sucrose, offers a promising substitute, yet clinical data in patients with MASLD are lacking. In a double-blind, randomized, two-period crossover trial, 103 adults with MASLD tasted iso-sweet vanilla ice cream sweetened with either brazzein or sucrose on two consecutive days. Overall impression and sensory attributes (appearance, color, aroma, taste, and texture) were rated on 5-point hedonic scales, and the percentage of the 100 g portion consumed was recorded. Brazzein-sweetened ice cream met the prespecified criteria for both non-inferiority and equivalence versus sucrose for overall impression. Top-2 box acceptance (ratings ≥ 4) was extremely high and nearly identical (96.1% for brazzein and 98.1% for sucrose). Mean consumption exceeded 98% of the portion for both products, with no significant difference between sweeteners. Secondary sensory ratings were closely similar, and multivariate analyses indicated highly overlapping sensory profiles. Exploratory subgroup analyses suggested consistent findings across most demographic and clinical characteristics, although participants with advanced liver fibrosis (LSM ≥ 9.6 kPa) showed numerically higher ratings for sucrose. In exploratory analyses, liver stiffness was associated with slightly lower intake at higher stiffness values. This study provides the first evidence that brazzein-sweetened ice cream maintains short-term sensory acceptability comparable to a conventional sucrose-sweetened product in adults with MASLD. These findings support further development and evaluation of brazzein-containing sugar-reduced foods, including repeated-exposure sensory studies and separate metabolic investigations. Full article

The objectives of this study were to evaluate the effects of different dietary omega-6 to omega-3 (n-6:n-3) fatty acid ratios during timed artificial insemination (TAI) on reproductive responses, luteal development, and offspring characteristics in lactating dairy cows. Holstein cows (n = 60) averaging 109 ± 10 days in milk (DIM) were randomly assigned to receive either a greater n-6:n-3 diet or a lower n-6:n-3 diet for 42 days. All cows were synchronized using a modified Ovsynch protocol. Although synchronized ovulation rates and pregnancy outcomes were not significantly affected by treatment, cows fed the lower n-6:n-3 ratio exhibited significantly greater corpus luteum (CL) volume and cross-sectional area on days 11 and 14 post-TAI, indicating enhanced luteal development. Pregnancy rates following the first AI and cumulative AI, as well as the number of services per conception, did not differ between treatments. However, cows fed the lower n-6:n-3 ratio tended to produce a greater proportion of female offspring (66.67% vs. 42.90%; p = 0.09). In addition, calves born to cows receiving the lower n-6:n-3 ratio had greater birth weights than calves born to cows fed the greater n-6:n-3 ratio diet. Additionally, the lower n-6:n-3 diet tended to increase milk yield and significantly increased lactose and solids-not-fat yields. In conclusion, a lower dietary n-6:n-3 ratio during a TAI program enhanced luteal development, tended to increase the proportion of female offspring and increased calf birth weight. These findings suggest that reducing the dietary n-6:n-3 ratio during the breeding period may enhance luteal development and may influence offspring sex ratio and calf birth weight in lactating dairy cows. Full article

Milk flow dynamics during mechanical milking are strongly influenced by species-specific mammary anatomy, milk partitioning between cisternal and alveolar compartments, and milking management. The present study aimed to compare milkability traits across the main dairy species reared in central Italy using a large dataset collected over 20 years. A total of 7315 animals were included: dairy cows (1103), buffaloes (2870), goats (2399), sheep (754), and donkeys (189). Milk flow curves were recorded using a portable Lactocorder^® device. The following traits were analyzed: milk yield (MY), lag time (LT), milk ejection time (MET), total milking time (TMT), peak flow rate (PFR), average flow rate (AFR), plateau phase (PL), bimodal phase (BM), and bimodality incidence (Bimo). Marked interspecific differences emerged. Dairy cows showed the highest MY and PFR, with bimodality occurring in 23.7% of curves. Buffaloes exhibited lower flow rates, prolonged LT, and extended TMT, reflecting their strong dependence on oxytocin-mediated alveolar milk ejection. Sheep demonstrated short milking times and low bimodality (13.5%), consistent with their large cisternal milk fraction. Goats displayed breed-dependent variability, with specialized dairy breeds showing higher PFR and longer TMT. Donkeys produced low milk volumes but exhibited rapid and efficient milk flow, with the lowest incidence of bimodality (7.4%). Overall, milk flow patterns reflected species-specific udder morphology and physiological mechanisms of milk ejection. Although this field-based study faces inherent limitations in environmental and protocol standardization across farms, the resulting long-term dataset remains highly representative. These findings highlight the importance of tailoring milking machine settings and prestimulation protocols to species and breed characteristics to optimize milking efficiency, labor management, and animal welfare. Full article

The increasing demand for sustainable and health-oriented dairy and alternative products has enhanced interest in reducing or modifying fat in cheese systems. However, such modifications often lead to undesirable changes in texture and flavor, highlighting the multifunctional roles of milk fat within the cheese matrix. Rather than serving solely as a compositional component, milk fat contributes fundamentally to structure organization and flavor development through its physicochemical properties, interactions with the protein network, and lipid-derived pathways. This review examines these roles from a mechanistic perspective and evaluates emerging lipid structuring approaches for texture modulation, while also discussing complementary approaches with potential to address flavor attributes. Collectively, it provides insights for rational formulation and guides future research toward the design of improved dairy and alternative cheese products. Full article

Heat stress (HS) alters rumen function and may compromise fermentation efficiency in dairy cows. This in vitro study evaluated the effects of six additives—betaine, sodium bicarbonate, bentonite clay, protected fat, Saccharomyces cerevisiae yeast, and Melissa officinalis (lemon balm)—on ruminal fermentation under increasing incubation temperatures (39.0 °C, 40.5 °C, 41.5 °C). This study was designed as an initial in vitro screening approach aimed at evaluating additive resilience under a conservative, worst-case thermal challenge. The highest incubation temperature (41.5 °C) was not used to reproduce the exact physiological rumen environment but rather served to identify additives capable of maintaining fermentation when exposed to extreme conditions that may occur during severe heat load. These results therefore constitute a preliminary step before in vivo validation under realistic HS scenarios. Fermentation was assessed after 24 h by measuring gas production, pH, redox potential, volatile fatty acids (VFAs), and ammonia (NH₃) and performing protozoa counts. Temperature alone produced limited effects in the control, with numerical increases in gas, acetate, total VFAs, and NH₃ at 41.5 °C. Betaine and yeast maintained overall stable fermentation across all temperatures, preserving gas production, pH, redox potential, and VFA profiles. Sodium bicarbonate increased the pH but reduced gas production. Lemon balm enhanced VFAs and propionate at 39.0 °C but showed reduced activity at high temperature. Protected fat and bentonite clay resulted in lower gas production with minimal influence on other parameters. The protozoa counts were not affected by temperature and showed statistically detectable differences among additives. Overall, betaine and yeast exhibited the highest thermal stability and appear to be suitable candidates for inclusion in feeding strategies aimed at supporting rumen fermentation during periods of HS. Full article

Ketosis is one of the most economically significant metabolic disorders affecting periparturient dairy cows, causing production losses and predisposing animals to secondary complications. Current blood-based diagnostics are invasive and provide limited insight into the underlying metabolic perturbations. This study employed an integrated three-platform metabolomics approach to characterize milk metabolite alterations in ketotic Holstein dairy cows and to evaluate milk-based biomarker panels for early ketosis detection. Milk samples from 20 healthy control (CON) cows and 6 ketotic cows were collected at 2 weeks postpartum and analyzed by direct injection/liquid chromatography–tandem mass spectrometry (DI/LC-MS/MS), proton nuclear magnetic resonance (¹H-NMR) spectroscopy, and inductively coupled plasma mass spectrometry (ICP-MS). Ketosis was confirmed by serum β-hydroxybutyrate concentrations ≥ 1400 μmol/L. Principal component analysis, partial least squares-discriminant analysis, and receiver operating characteristic (ROC) curve analyses were applied. All three platforms discriminated ketotic cows from healthy cows, with clear cluster separation validated by 2000 permutation tests (p < 0.05). DI/LC-MS/MS identified 16 significantly altered metabolites (p < 0.05), with butyrylcarnitine (C4), phosphatidylcholine 30:0 (PC 30:0), ether-linked phosphatidylcholine O-38:3 (PC O-38:3), and citrulline identified as the top discriminatory biomarkers (AUC = 0.920; 95% CI: 0.85–0.98; sensitivity = 91.7%; specificity = 93.3%). ICP-MS revealed significantly reduced selenium (Se, p = 0.017), manganese (Mn, p = 0.045), and chromium (Cr, p = 0.037), as well as elevated cobalt (Co, p = 0.014) in ketotic milk (AUC = 0.870). ¹H-NMR detected no individually significant metabolites; however, multivariate analysis distinguished groups (AUC = 0.890), with succinate (numerical fold change: +5.77×; p = 0.059), methanol (−1.94×; not significant), and acetate (+2.88×; not significant) as top VIP contributors. The combined multi-platform biomarker panel (joint classification using top VIP features from all three platforms, without formal data fusion) achieved superior diagnostic performance (AUC = 0.970; 95% CI: 0.93–1.00; sensitivity = 95.0%; specificity = 96.7%). These findings identify coordinated perturbations in glycerophospholipid metabolism, acylcarnitine profiles, amino acid homeostasis, antioxidant mineral status, and energy metabolism during early ketosis, and suggest that milk metabolomics is a promising non-invasive approach for precision dairy health monitoring, pending validation in independent cohorts. We acknowledge the small ketotic group size (n = 6) as a limitation; therefore, these findings should be considered discovery cohort observations requiring prospective validation before clinical translation. Full article

Genetic screening of monogenic traits is important for improving genetic health and increasing favorable milk protein in dairy cattle. This study developed and applied an amplification refractory mutation system PCR (ARMS-PCR) assay to simultaneously screen 21 causal variants underlying 18 monogenic traits in Holstein cattle, including 13 recessive genetic defects, 2 milk protein loci, and 3 morphological loci. The assay was designed as a unified multiplex PCR-capillary electrophoresis workflow, enabling clear detection of allele-specific products distinguished by fragment size and fluorescent color. Sanger sequencing validation of newly incorporated loci supported the accuracy of the assay. A total of 1656 cows from 12 commercial farms were genotyped using the multiplex ARMS-PCR panel, and amplicons were analyzed by capillary electrophoresis. Carriers were detected for all genetic defects except DUMPS, with carrier frequencies ranging from 0.12% to 6.64%. The highest frequencies were observed for HH5 (6.64%) and MWS (6.58%), whereas HH3, HH1, and HCD showed intermediate frequencies of 1.81% to 3.08%; all the remaining defects were below 1%. Overall, 22.10% of sampled cows carried at least 1 defect allele, including 20.47% carrying 1 defect, 1.51% carrying 2, and 0.12% carrying 3. For milk protein loci, the desirable β-casein A2A2 and κ-casein BB genotypes occurred at frequencies of 45.83% and 14.13%, respectively, and the favorable A2A2/BB combination was present in 6.22% of sampled cows. Dominant red, recessive red, and polled alleles were rare. These results indicate that multiplex fluorescent ARMS-PCR can serve as a practical targeted screening tool for the simultaneous management of known deleterious alleles and selection of favorable monogenic variants in dairy cattle breeding. Full article

Milk adulteration is a common form of food fraud, particularly in high-value dairy products from small ruminants. A frequent practice involves dilution with water, often combined with the addition of sugars to mask physicochemical changes and avoid detection during routine quality control. This study aimed to develop an analytical approach for detecting combined adulteration in goat and sheep milk involving both water dilution and sucrose addition. Controlled experiments were conducted by diluting milk samples with water (1–15%) followed by the addition of sucrose solutions. Changes in physicochemical parameters, including fat, protein, total solids, lactose, density, freezing point depression, mineral content, and pH, were evaluated using an automated milk analyzer. In parallel, a suspected adulterant powder was characterized using conventional chemical analysis, ICP-AES, and HPLC-RI, revealing a composition predominantly of sucrose (91.4% w/w) with elevated sodium levels. Sucrose in milk samples was subsequently quantified using an enzymatic spectrophotometric method. Water dilution reduced protein, total solids, and density, while sucrose addition partially restored these parameters, masking adulteration effects. However, sucrose was reliably detected at concentrations above 0.1%. The proposed workflow may provide a practical and cost-effective complementary tool for routine dairy authenticity surveillance and fraud prevention. Full article