This research paper addresses the hypotheses (1) that milk produced from hay-fed cows differs from that of silage-fed cows and (2) that silage type has an important impact, too. Four diets differing in forage type but with equal estimated milk production potential and a forage:concentrate ratio of 0.85 : 0.15 were compared regarding their effect on feed intake, milk yield and milk properties. The forages tested were hay, grass silage, conventional short-chopped and long-chopped maize silage subjected to a novel processing technology (Shredlage®). Twenty-four dairy cows were fed two of the four diets in two consecutive runs in an incomplete (4 × 2) Latin-square design (n = 12 per diet). Each experimental period lasted 22 d, with 12 d of adaptation and 10 d of sampling. During sampling, feed intake and milk yield were recorded daily, milk composition and coagulation properties were determined four times. The composition of the diet ingredients was analysed weekly. Data were analysed with a mixed model considering feed, period and their interaction as fixed effects. Stage of lactation, milk yield and milk composition from the pre-experimental period were used as covariates in the model. Dry matter intake was lower with the long-chopped processed maize silage compared to the other three groups. There were some diet differences in intakes of net energy for lactation and absorbable protein in the duodenum, but this did not result in changes in milk yield. The milk fat content was higher with the grassland-based diets compared to the maize silage diets. No treatment effect on milk acidity and rennet coagulation properties was observed. In conclusion, there were no indications for specific physico-chemical properties of milk from a hay-based diet, and maize processing technology was not of large effect either. Future investigations should focus on sensory differentiation of the milk produced with different forages.

Lipopolysaccharides (LPS) could induce milk fat depression via regulating the body and blood fat metabolism. However, it is not completely clear how LPS might regulate triglyceride synthesis in dairy cow mammary epithelial cells (DCMECs). DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS. The level of triglyceride synthesis, the expression and activity of the liver X receptor α (LXRα), enzymes related to de novo fatty acid synthesis, and the expression of the fatty acid transporters were investigated. We found that LPS decreased the level of triglyceride synthesis via a down-regulation of the transcription, translation, and nuclear translocation level of the LXRα. The results also indicated that the transcription level of the LXRα target genes, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthetase (FAS), acetyl-CoA carboxylase-1 (ACC1), were significantly down-regulated in DCMECs after LPS treatment. Our data may provide new insight into the mechanisms of milk fat depression caused by LPS.

The aim of the work reported here was to investigate the appropriateness of a sinusoidal function by applying it to model the cumulative lactation curves for milk yield and composition in primiparous Holstein cows, and to compare it with three conventional growth models (linear, Richards and Morgan). Data used in this study were 911 144 test-day records for milk, fat and protein yields, which were recorded on 834 dairy herds from 2000 to 2011 by the Animal Breeding Centre and Promotion of Animal Products of Iran. Each function was fitted to the test-day production records using appropriate procedures in SAS (PROC REG for the linear model and PROC NLIN for the Richards, Morgan and sinusoidal equations) and the parameters were estimated. The models were tested for goodness of fit using adjusted coefficient of determination $\lpar {R_{{\rm adj}}^2 } \rpar $, root mean square error (RMSE), Akaike's information criterion (AIC) and the Bayesian information criterion (BIC). $R_{{\rm adj}}^2 $ values were generally high (>0.999), implying suitable fits to the data, and showed little differences among the models for cumulative yields. The sinusoidal equation provided the lowest values of RMSE, AIC and BIC, and therefore the best fit to the lactation curve for cumulative milk, fat and protein yields. The linear model gave the poorest fit to the cumulative lactation curve for all production traits. The current results show that classical growth functions can be fitted accurately to cumulative lactation curves for production traits, but the new sinusoidal equation introduced herein, by providing best goodness of fit, can be considered a useful alternative to conventional models in dairy research.

This Research Reflection short review will discuss vitamin D metabolism, its role in nutrition, disease prevention, and welfare of dairy cattle, as well as its toxicity. Vitamin D is an important fat-soluble vitamin. However, some researchers regard it as a hormone due to its function in the organism. Its role is not limited just to Ca homoeostasis and bone metabolism but is also associated with immunity. In dairy cattle it is known for preventing milk fever. Cows can acquire vitamin D in many ways for example through feed, parenteral injections or through UVB irradiation from the sun or artificial lighting. The vitamin D in feed can either be plant-/ fungi- based ergocalciferol or animal-based cholecalciferol. There is currently only one registered feed vitamin D supplement for cattle in the European Union and it is cholecalciferol. Animals can also synthesize their own vitamin D when 7-dihydrocholesterol in the skin is irradiated with UVB light resulting in cholecalciferol production. Despite its importance, many cattle are deficient in vitamin D due to inadequate supplementation or insufficient sun exposure. In a study performed at the Veterinary Faculty in Slovenia 12 high producing Holstein Friesian cows at a commercial dairy farm were blood tested for vitamin D status for three succeeding months and all but one were vitamin D insufficient in all testings. The cows were not exposed to direct sunlight and the content of vitamin D3 in feed was <400 IU/kg dry matter, which is less than half of the NRC (2001) recommendation. Deficiency can also occur due to diseases affecting the gastrointestinal tract, such as paratuberculosis, which lower the absorptive capacity of the gut. Vitamin D can be toxic if cows are over-supplemented or consume large quantities of plants like Trisetum flavescens, which contain an active form of vitamin D-calcitriol or its glycosides, that are activated by digestion in the rumen.

The experiments reported in this research paper aimed to determine the effect of supplementing different forms of L-methionine (L-Met) and acetate on protein synthesis in immortalized bovine mammary epithelial cell line (MAC-T cells). Treatments were Control, L-Met, conjugated L-Met and acetate (CMA), and non-conjugated L-Met and Acetate (NMA). Protein synthesis mechanism was determined by omics method. NMA group had the highest protein content in the media and CSN2 mRNA expression levels (P < 0.05). The number of upregulated and downregulated proteins observed were 39 and 77 in L-Met group, 62 and 80 in CMA group and 50 and 81 in NMA group from 448 proteins, respectively (P < 0.05). L-Met, NMA and CMA treatments stimulated pathways related to protein and energy metabolism (P < 0.05). Metabolomic analysis also revealed that L-Met, CMA and NMA treatments resulted in increases of several metabolites (P < 0.05). In conclusion, NMA treatment increased protein concentration and expression level of CSN2 mRNA in MAC-T cells compared to control as well as L-Met and CMA treatments through increased expression of milk protein synthesis-related genes and production of the proteins and metabolites involved in energy and protein synthesis pathways.

In this research communication we describe the creation of an equation for the prediction of milk yield (MY) from udder volume (UV). A total of 280 measurements were collected between 5 and 15 d postpartum (pp) from 36 multiparous Pelibuey ewes. Study variables were measured between 2 and 9 weeks pp and MY was measured by manual milking, UV prior to and following milking was measured using the technique of making moulds from aluminium foil. The MY ranged from 0.09 to 0.83 kg/d, meanwhile UV prior and following milking ranged from 155 to 1940 and 90 to 1520 cm3, respectively. Measurements of UV had a moderate to high (P < 0.01; 0.58 ≤ r ≤ 0.78) correlation with MY. The UV prior to milking was the best prediction model for MY, which explained 62% of the variation in MY. This equation presented moderate precision (r2 = 0.61) and high accuracy (bias correction factor = 0.94), confirming a good reproducibility index (concordance correlation coefficient = 0.73). Modelling efficiency (MEF = 0.59) showed moderate concordance between observed and predicted values. In conclusion, MY in lactating Pelibuey ewes could be predicted in a moderate way using the predictor variable UV measured with the technique of moulds made with aluminium foil.

In this Research Reflection I shall develop and validate the hypothesis that lactation insufficiency in obese breastfeeding mothers has an endocrine explanation. I shall not present data, but I shall review pertinent literature to show that obesity is associated with a partial or sometimes complete failure to initiate and maintain lactation, and critically examine the belief that this is due to psychosocial factors, a failure of prolactin secretion or both. Since progesterone is inhibitory to lactogenesis and oestrogens are inhibitory to milk secretion, I shall then explore the possibility that these steroids are linked to lactation failure, through sequestration of progesterone and aromatization of oestrogen in mammary adipose tissue. I shall conclude by describing experimental approaches in animal models that could be used to test this hypothesis.

In Brazil, the buffalo milk market has been growing. However, identity and quality standards have not been established for this raw material, nor have proper distinctions between buffalo milk and bovine milk been defined. Currently, the State of Rio Grande do Sul (RS) has only three producers that supply raw material for officially marketed derivatives. The aim of this study was to determine the identity and quality standards of raw buffalo milk in this region. Samples were obtained biweekly from three farm cooling tanks between June 2017 and August 2018, to reach a total of 69 samples. The averages for the results of the physicochemical parameters fat, protein, lactose, total solids, SNF (solids-not-fat), calcium, density, FP, acidity and SCC were 5.5 g/100 g, 4.06 g/100 g, 5.07 g/100 g, 15.5 g/100 g, 9.96 g/100 g, 0.161 g/100 g, 1.034 g/ml, −0.527°C, 16°D and 95 × 103 cells/ml, respectively. With reference to the microbiological parameters, the mean of the Standard Plate Count (SPC) and thermotolerant coliforms were 9,0 × 104 CFU/ml and 1.6 × 102 MPN/ml, respectively. Regarding coagulase-positive staphylococci, 36 samples tested positive (52% of total). Neither Salmonella spp. nor Listeria monocytogenes, nor antibiotic or antiparasitic residues were detected in any sample. In conclusion, the buffalo milk used as raw material for dairy products in southern Brazil demonstrated satisfactory physicochemical and microbiological characteristics, in accordance with recent scientific literature.

In this research communication, a cell model with elevated β-CASEIN synthesis was established by stimulating bovine mammary epithelial cells with 0.6 mM methionine, and the genome-wide gene expression profiles of methionine-stimulated cells and untreated cells were investigated by RNA sequencing. A total of 458 differentially expressed genes (DEGs; 219 upregulated and 239 downregulated) were identified between the two groups. Gene Ontology (GO) analysis showed that the two highest-ranked GO terms in ‘molecular function’ category were ‘binding’ and ‘catalytic activity’, suggesting that milk protein synthesis in methionine-stimulated cells requires induction of gene expression to increase metabolic activity. Kyoto Encyclopedia of Genes and Genomes analysis revealed that within the ‘environmental information processing’ category, the subcategory that is most highly enriched for DEGs was ‘signal transduction’. cGMP-PKG, Rap1, calcium, cAMP, PI3K-AKT, MAPK, and JAK-STAT are the pathways with the highest number of DEGs, suggesting that these signaling pathways have potential roles in mediating methionine-induced milk protein synthesis in bovine mammary epithelial cells. This study provides valuable insights into the physiological and metabolic adaptations in cells stimulated with methionine. Understanding the regulation of this transition is essential for effective intervention in the lactation process.

It is common practice in the dairy industry to separate the calf from the cow immediately after parturition, and in most parts of the world calves are housed individually during the milk-feeding period. Early and abrupt separation has major implications for the calf's physical and psychological development. In this Research Reflection short review we present and discuss the main housing systems and management practices regarding early weaning in today's dairy industry. Main benefits and disadvantages are critically addressed, and possible future research suggested. Furthermore, major policy issues related to consumers, scientific recommendations and economic performance of farms have been identified, as well as future drivers for more viable housing solutions for neonatal calves. This review serves as an introduction and preamble to the second section of this Special Issue, which is dedicated to cow-calf contact management systems.

The aims of the research reported here were to identify potential risk factors associated with the presence of Staphylococcus aureus intramammary infection (IMI) in pre partum dairy heifers on 17 dairy farms from three provinces of Argentina and to characterize, at molecular level, isolates from those heifers and lactating cows from two selected herds. A total of 1474 heifers and 4878 lactating cows were studied. The prevalence of Staphylococcus aureus IMI in the heifers, heifers at quarter level and lactating cow mammary quarters was 14.41, 4.82, and 14.65%, respectively. Univariate analysis showed the key variables associated with S. aureus IMI presence in the heifers were: S. aureus IMI prevalence in cows of the lactating herd, the time calves stayed with their dam after birth, the calf rearing system, the place of rearing (own farm or other dairy farm) and fly control on the farm. None of the variables included in the multivariable analysis was associated with the presence of S. aureus IMI in the pre partum heifers, probably due to low variability among management practices used by the farms for rearing the heifer calves. At the molecular level, S. aureus isolates were grouped into three main PFGE clusters and several genotypes within the clusters. Isolates from mammary secretion of pre partum heifers and milk of lactating cows comprised different PFGE clusters in both herds, although two exceptions occurred. The absence of gene fnbpB, which codifies for a virulence factor protein involved in cell invasion by S. aureus, was significantly more frequent in pre partum heifer secretion isolates than in isolates from lactating cow milk. These results suggest that, under these management conditions, isolates from mammary secretions of pre partum heifers do not originate from the milk of lactating cows, but rather other sources to which the heifer is exposed.

In this Research Reflection we review management practices in small family farms with less than 100 cows. Small farms represent the majority of farms in the EU and the world, and they are of great importance for the economy of a country. On cattle farms, the welfare of calves is of primary importance for the profitability of the herd, and poor management is one of the main factors influencing calf health and survival. Data on the risk factors for calf welfare issues in small-scale farms are limited. For this purpose, the literature data from six world countries were presented and compared, including Serbia and Slovenia where a survey related to the issue was carried out within the COST Action FA1308, DairyCare. Some practices within the following areas in calf management were considered: calving management, care for new-born calves, use of painful procedures, colostrum management, cow–calf separation, calf feeding, weaning, calf housing, and general monitoring. In each of the countries, the health and welfare of calves are threatened by some omissions in rearing practices and the major are related to the new-born calf management, the feeding and watering management, and the application of hygienic measures. Many farmers are well aware of the importance of proper calf rearing; others would need more incentive to improve calf management. Each country should pay attention to the education of farmers about the most common deficiencies in calf management.

This study aimed to compare the effect of different ratios of Streptococcus thermophilus to Lactobacillus bulgaricus (3 : 1, 1 : 1, and 1 : 3) under the various stressful temperatures (37 and 45°C) on the fatty acid profiles quality of Kermanshahi roghan (yogurt by-product) and sour cream to obtain a formula for producing a kind of animal fat healthier than milk and cream. Stresses such as fermentation play an important role in bacterial behavior and consequently in food quality. Our findings presented a significant difference between roghan and sour cream fatty acid levels only at 37°C. Furthermore, starter culture 3 : 1 was the best starter for producing products with a higher quality of fatty acid profile at 37°C, and a 1 : 1 S. thermophilus to L. bulgaricus ratio was optimal at 45°C. It seems that bacteria adapt to harsh growth conditions by changing the fatty acid profiles, and these changes warrant consideration in the production of a kind of animal fat with the best fatty acid profiles. In conclusion, the roghan fatty acid profile is more suitable than sour cream only at 37°C.

In this research paper we filter and verify miRNAs which may target silent information regulator homolog 2 (SIRT2) gene and then describe the mechanism whereby miRNA-212 might regulate lipogenic genes in mammary epithelial cell lines via targeting SIRT2. Bioinformatics analysis revealed that the bovine SIRT2 gene is regulated by three miRNAs: miR-212, miR-375 and miR-655. The three miRNAs were verified and screened by qRT-PCR, western blot, and luciferase multiplex verification techniques and only miR-212 was shown to have a targeting relationship with SIRT2. The results of co-transfecting miR-212 and silencing RNA (siRNA) showed that by targeting SIRT2, miR-212 can regulate the expression of fatty acid synthetase (FASN) and sterol regulatory element binding factor 1 (SREBP1) but not peroxisome proliferator-activated receptor gamma (PPARγ). Measurement of triglyceride (TAG) content showed that miR-212 increased the fat content of mammary epithelial cell lines. The study indicates that miR-212 could target and inhibit the expression of the SIRT2 gene to promote lipogenesis in mammary epithelial cell lines.

We determined the effects of a combination of C18 unsaturated fatty acids (C18-UFAs) consisting of oleic, linoleic, and linolenic acids on milk lipogenesis in bovine mammary epithelial cells (BMECs). By orthogonal experiments to determine cellular triacylglycerol (TAG) accumulation, a combination of 200 μmol/l C18 : 1, 50 μmol/l C18 : 2, and 2 μmol/l C18 : 3 was selected as C18-UFAs combination treatment, and culture in medium containing fatty acid-free bovine serum albumin was used as the control. The expression of genes related to milk lipid synthesis and intracellular FA composition was measured. The results showed that cytosolic TAG formation was higher under C18-UFAs treatment than under control treatment. The mRNA expression of acetyl-CoA carboxylase-α (ACACA), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor gamma (PPARG) did not differ between treatments. The abundance of stearoyl-CoA desaturase (SCD) and acyl-CoA synthetase long-chain family member 1 (ACSL1) was higher, whereas that of sterol regulatory element binding transcription factor 1 (SREBF-1) was lower after C18-UFAs treatment compared to control treatment. The C16 : 0 and SFA content was decreased following C18-UFAs treatment compared to control treatment, while the cis-9 C18 : 1 and UFA content was increased. In conclusion, C18-UFAs could stimulate triglyceride accumulation, increase the cellular UFA concentration, and regulate lipogenic genes in BMECs.

Traditional Mozzarella is a fresh cheese produced in Italian local market without additives that shows a short shelf life of about 5 d. This work tested the use of natural additives (bergamot juice concentrate-BJ and calcium lactate-CL) in preserving liquid for a Mozzarella cheese with the aim to extend its shelf life, regarding the microbial growth and overall cheese quality. Results of qualitative analyses showed that the preserving liquid with the mix of BJ and CL promoted an extension of mozzarella shelf life up to 20 d. A slightly reduced growth of Pseudomonas species was evidenced after 5 d of storage, whereas no inhibition of lactic acid bacteria was observed for the storage period. Moreover, mozzarella cheese packed in mixed preserving liquid possessed better textural properties, evidenced by the lowest proteolysis index measured after 13 d of storage, and a good antioxidant activity.

The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and β-lactoglobulin as well as αs1- and β-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.

In this research communication we describe an innovative protocol that combines three pairs of primers, two from the literature and one designed in our laboratory, for application in triplex-PCR on somatic cell DNA to enable identification of the species origin (cow, sheep, goat) of cheeses and yogurts with a detection limit of 0.1%. Mislabeling was detected in 15 out of 40 cheeses and in 18 out of 40 yogurts tested. The suggested procedure is a quick and reliable tool for identifying the animal origin of cheeses and yogurts and it can be used to certify product reliability on the domestic and international market. Additionally, in combination with a serological test it can offer a reliable tool for detecting the presence of cow's whey.

We hypothesized that insulin-induced gene 1 (INSIG1) affects milk fat synthesis in buffalo. For this reason, the protein abundance of INSIG1 in the mammary tissue of buffalo during the peak period of lactation and dry-off period was evaluated. The results showed that the expression of INSIG1 at the peak of lactation was lower than that in the dry-off period. To explore the role of INSIG1 in milk fat synthesis, the buffalo mammary epithelial cells (BMECs) were isolated and purified from buffalo mammary tissue, and INSIG1 gene were overexpressed and knocked down by constructing the recombinant lentivirus vector of INSIG1 gene and transfecting into BMECs. Results revealed that INSIG1 overexpression decreased the expression of INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6, which led to reduction of triglycerides (TAG) content in the cell. In contrast, knockdown of INSIG1 had a positive effect on mRNA expression of the above genes. Overall, the data provide strong support for a key role of INSIG1 in the regulation of milk fat synthesis in BMECs.