Shifts in the Holstein dairy cow milk fat globule membrane proteome that occur during the first week of lactation are affected by parity.

BACKGROUND: The milk fat globule membrane (MFGM) proteomes of colostrum and transition milk are rich sources of proteins that are likely important for neonatal calf health. In addition, characterization of these proteomes could also yield valuable information regarding mammary gland physiology of the early postpartum lactating cow. The objectives of this research were to characterize the MFGM proteomes of colostrum and transition milk through sample collections at four timepoints postpartum, including the first milking (M1, colostrum), second milking (M2, transition milk), fourth milking (M4, transition milk), and fourteenth milking (M14, mature milk), and compare these proteomes between multiparous (MP; n = 10) and primiparous (PP; n = 10) Holstein dairy cows. Isolated MFGM proteins were labeled using Tandem Mass tagging and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification was completed using MASCOT and Sequest in Proteome Discoverer 2.2. The scaled abundance values were analyzed using PROC MIXED in SAS to determine the effects of milking (MIL), parity (PAR), and MIL × PAR. The adaptive false-discovery rate (FDR)-adjusted P values were determined using PROC MULTTEST. Protein characterization and bioinformatic analysis were completed using a combination of PANTHER, Blast, and Uniprot. RESULTS: A total of 104 common proteins were identified in each of the MFGM samples. Statistical analysis revealed that 70.2% of identified proteins were affected by MIL. Of these, 78.1% were lower in M14 compared with M1, including immune-related proteins lactotransferrin, lactadherin and hemopexin. Parity affected 44.2% of proteins. Of the proteins affected by PAR, 84.8% were higher in MP cows compared with PP cows, including apolipoprotein E and histones 2A, 2B, 3, and 4 b. Butyrophilin subfamily 1 member 1A and annexin 5 were higher in samples from PP cows. Milking × parity affected 32.7% of identified proteins, including lactotransferrin, gelsolin, vitamin D binding protein, and S100 proteins. CONCLUSIONS: This research supports previous findings that the Holstein MFGM proteome changes rapidly during the first week of lactation. In addition, this research identifies the impact of parity on the colostrum and transition milk MFGM proteomes, which may be important for milk-fed calf health or for the identification of protein biomarkers for mammary functionality.

Characterization of variations within the rumen metaproteome of Holstein dairy cattle relative to morning feed offering.

Few studies have utilized proteomic techniques to progress our knowledge of protein-mediated pathways within the rumen microbial community, and no previous research has used these techniques to investigate the patterns or variations of these proteins within this community. It was hypothesized that there would be fluctuations of rumen microbial protein abundances due to feed intake-mediated nutrient availability and that these could be identified using non gel-based proteomic techniques. This study investigated the fluctuations of bovine rumen metaproteome utilizing three mid to late-lactation Holsteins. Rumen fluid was collected at three timepoints on three days relative to their first morning feed offering (0 h, 4 h, and 6 h). Samples were pooled within timepoint within cow across day, analyzed using LC-MS/MS techniques, and analyzed for variations across hour of sampling using PROC MIXED of SAS with orthogonal contrasts to determine linear and quadratic effects. A total of 658 proteins were characterized across 19 microbial species, with 68 proteins identified from a variety of 15 species affected by time of collection. Translation-related proteins such as 50S and 30S ribosomal protein subunit variants and elongation factors were positively correlated with hour of sampling. Results suggest that as nutrients become more readily available, microbes shift from conversion-focused biosynthetic routes to more encompassing DNA-driven pathways.

Inclusion of grape marc in dairy cattle rations alters the bovine milk proteome.

Grape marc (GPM) is a viticulture by-product that is rich in secondary compounds, including condensed tannins (CT), and is used as a supplement in livestock feeding practices. The aim of this study was to determine whether feeding GPM to lactating dairy cows would alter the milk proteome through changes in nitrogen (N) partitioning. Ten lactating Holstein cows were fed a total mixed ration (TMR) top-dressed with either 1.5 kg dry matter (DM)/cow/day GPM (GPM group; n = 5) or 2.0 kg DM/cow/day of a 50:50 beet pulp: soy hulls mix (control group; n = 5). Characterization of N partitioning and calculation of N partitioning was completed through analysis of plasma urea-N, urine, feces, and milk urea-N. Milk samples were collected for general composition analysis, HPLC quantification of the high abundance milk proteins (including casein isoforms, α-lactalbumin, and ß-lactoglobulin) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the low abundance protein enriched milk fraction. No differences in DMI, N parameters, or calculated N partitioning were observed across treatments. Dietary treatment did not affect milk yield, milk protein or fat content or yield, or the concentrations of high abundance milk proteins quantified by HPLC analysis. Of the 127 milk proteins that were identified by LC-MS/MS analysis, 16 were affected by treatment, including plasma proteins and proteins associated with the blood-milk barrier, suggesting changes in mammary passage. Immunomodulatory proteins, including butyrophilin subfamily 1 member 1A and serum amyloid A protein, were higher in milk from GPM-fed cows. Heightened abundance of bioactive proteins in milk caused by dietary-induced shifts in mammary passage could be a feasible method to enhance the healthfulness of milk for both the milk-fed calf and human consumer. Additionally, the proteome shifts observed in this trial could provide a starting point for the identification of biomarkers suitable for use as indicators of mammary function.

Symposium review: Characterization of the bovine milk protein profile using proteomic techniques.

Identification and characterization of the comprehensive bovine milk proteome has historically been limited due to the dichotomy of protein abundances within milk. The high abundance of a select few proteins, including caseins, α-lactalbumin, ß-lactoglobulin, and serum albumin, has hindered intensive identification and characterization of the vast array of low-abundance proteins in milk due to limitations in separation techniques and protein labeling capacity. In more recent years, the development and advancement of proteomics techniques have yielded valuable tools for characterization of the protein profile in bovine milk. More extensive fractionation and enrichment techniques, including the use of combinations of precipitation techniques, immunosorption, gel electrophoresis, chromatography, ultracentrifugation, and hexapeptide-based binding enrichment, have allowed for better isolation of lower abundance proteins for further downstream liquid chromatography-tandem mass spectrometry approaches. The different milk subfractions isolated during these processes can also be analyzed as individual entities to assess the protein profile unique to the different fractions-for instance, investigation of the skim milk-associated proteome versus the milk fat globule membrane-associated proteome. Updates to high-throughput methods, equipment, and software have also allowed for greater interpretation and visualization of the data. For instance, labeling techniques have enabled analysis of multiplexed samples and more accurate comparison of specific protein abundances and quantities across samples, and integration of gene ontology analysis has allowed for a more in-depth and visual representation of potential relationships between identified proteins. Inclusively, these developments in proteomic techniques have allowed for a rapid increase in the number of milk-associated proteins identified and a better grasp of the relationships and potential functionality of the proteins within the milk proteome.

Comparative proteomics dataset of skimmed milk samples from Holstein and Jersey dairy cattle.

Milk samples were collected from Holstein and Jersey breeds of dairy cattle maintained under the same management practices and environmental conditions over a seven-day period. Milk samples were collected twice daily from six cows of each breed as previously described (Tacoma et al., 2016) [1]. Samples were composited within individual cow over the experimental period and skimmed to remove the fat layer. Skimmed milk samples were fractionated using CaCl2 precipitation, ultracentrifugation and ProteoMiner treatment to remove the high abundance milk proteins. Separation of the low abundance proteins was achieved using SDS-PAGE. Differential protein abundances were analyzed by mass spectrometry-based proteomic approaches followed by statistical analyses of the peptide count data. The complete list of low-abundance proteins identified in both breeds is provided in the dataset as well as the total number of distinct sequenced peptides and gene ontology functions for each protein. The relative abundance of a select few proteins is depicted using the SIEVE software.

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows.

Milk is a highly nutritious natural product that provides not only a rich source of amino acids to the consumer but also hundreds of bioactive peptides and proteins known to elicit health-benefitting activities. We investigated the milk protein profile produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions using proteomic approaches that optimize protein extraction and characterization of the low abundance proteins within the skim milk fraction of bovine milk. In total, 935 low abundance proteins were identified. Gene ontology classified all proteins identified into various cellular localization and function categories. A total of 43 low abundance proteins were differentially expressed between the two dairy breeds. Bioactive proteins involved in host-defense, including lactotransferrin (P=0.0026) and complement C2 protein (P=0.0001), were differentially expressed by the two breeds, whereas others such as osteopontin (P=0.1788) and lactoperoxidase (P=0.2973) were not. This work is the first to outline the protein profile produced by two important breeds of dairy cattle maintained under the same diet, environment and management conditions in order to observe likely true breed differences. This research now allows us to better understand and contrast further research examining the bovine proteome that includes these different breeds. BIOLOGICAL SIGNIFICANCE: Within the last decade, the amount of research characterizing the bovine milk proteome has increased due to growing interest in the bioactive proteins that are present in milk. Proteomic analysis of low abundance whey proteins has mainly focused on human breast milk; however, previous research has highlighted the presence of bioactive proteins in bovine milk. Recent publications outlining the cross-reactivity of bovine bioactive proteins on human biological function highlight the need for further investigation into the bovine milk proteome. The rationale behind this study is to characterize and compare the low abundance protein profile in the skim milk fraction produced from Holstein and Jersey breeds of dairy cattle, which are two major dairy cattle breeds in the USA. A combination of fractionation strategies was used to efficiently enrich the low abundance proteins from bovine skim milk for proteomic profiling. A total of 935 low abundance proteins were identified and compared between the two bovine breeds. The results from this study provide insight into breed differences and similarities in the milk proteome profile produced by two breeds of dairy cattle.

Effect of subacute ruminal acidosis on milk fat concentration, yield and fatty acid profile of dairy cows receiving soybean oil.

The objective of this study was to investigate the effect of ruminal infusion of soybean oil (SBO) with either a moderate- or high-forage diet on fat concentration, yield and composition in milk from dairy cows. Six rumen-fistulated Holstein dairy cows (639+/-51 kg body weight, 140+/-59 days in milk) were used in the study. Cows were randomly assigned to one of two dietary treatments, a high forage:concentrate (HFC, 74:26) or a moderate forage:concentrate (MFC, 56:44) total mixed ration. Cows were fed at 08.00 and 13.00 h and pulse-dosed ruminally at 13.00 h over a 10-min duration with 2% of diet dry matter of SBO. Ruminal pH was recorded continuously. Cows receiving the MFC treatment had lower daily mean ruminal pH and ruminal pH was below 6.0 for a longer duration compared with the HFC treatment (640 vs. 262 min/d, P<0.05). Cows receiving the MFC treatment had a greater reduction (diet by week interaction, P<0.05) in milk fat concentration and yield than cows receiving the HFC treatment (42 vs. 22% and 45 vs. 21%, respectively). Additionally, cows receiving the MFC diet had a greater reduction in milk fat concentration (g/100 g FA) of FA C16 (17 vs. 9%), trans-10 18:1 (159 vs. 21%) and trans-9, cis-11 conjugated linoleic acid (121 vs. 55%) (P<0.05) compared with cows receiving the HFC diet. This study demonstrated that cows fed the MFC diet had lower ruminal pH and showed a greater rate of milk fat depression when infused with SBO.