Ratio of dietary rumen degradable protein to rumen undegradable protein affects nitrogen partitioning but does not affect the bovine milk proteome produced by mid-lactation Holstein dairy cows

Abstract

Little is known about the bovine milk proteome or whether it can be affected by diet. The objective of this study was to determine if the dietary rumen degradable protein (RDP):rumen undegradable protein (RUP) ratio could alter the bovine milk proteome. Six Holstein cows (parity: 2.5 ± 0.8) in mid lactation were blocked by days in milk (80 ± 43 d in milk) and milk yield (57.5 ± 6.0 kg) and randomly assigned to treatment groups. The experiment was conducted as a double-crossover design consisting of three 21-d periods. Within each period, treatment groups received diets with either (1) a high RDP:RUP ratio (RDP treatment: 62.4:37.6% of crude protein) or (2) a low RDP:RUP ratio (RUP treatment: 51.3:48.7% of crude protein). Both diets were isonitrogenous and isoenergetic (crude protein: 18.5%, net energy for lactation: 1.8 Mcal/kg of dry matter). To confirm N and energy status of cows, dry matter intake was determined daily, rumen fluid samples were collected for volatile fatty acid analysis, blood samples were collected for plasma glucose, β-hydroxybutyrate, urea nitrogen, and fatty acid analysis, and total 24-h urine and fecal samples were collected for N analysis. Milk samples were collected to determine the general milk composition and the protein profile. Milk samples collected for high-abundance protein analysis were subjected to HPLC analysis to determine the content of α-casein, β-casein, and κ-casein, as well as α-lactalbumin and β-lactoglobulin. Samples collected for low-abundance protein analysis were fractionated, enriched using ProteoMiner treatment, and separated using sodium dodecyl sulfate-PAGE. After excision and digestion, the peptides were analyzed using liquid chromatography (LC) tandem mass spectrometry (MS/MS). The LC-MS/MS data were analyzed using PROC GLIMMIX of SAS (version 9.4, SAS Institute Inc., Cary, NC) and adjusted using the MULTTEST procedure. All other parameters were analyzed using PROC MIXED of SAS. No treatment differences were observed in dry matter intake, milk yield, general milk composition, plasma parameters, or rumen volatile fatty acid concentrations, indicating no shift in total energy or protein available. Milk urea N and plasma urea N concentrations were higher in the RDP group, indicating some shift in N partitioning due to diet. A total of 595 milk proteins were identified, with 83% of these proteins known to be involved in cellular processes. Although none of the low-abundance proteins identified by LC-MS/MS were affected by diet, feeding a diet high in RUP decreased β-casein, κ-casein, and total milk casein concentration. Further investigations of the interactions between diet and the milk protein profile are needed to manipulate the milk proteome using diet.

Validating and optimizing spot sampling of urine to estimate urine output with creatinine as a marker in dairy cows

“…However, N intake, urinary N excretion, and urinary N excretion as proportion of N intake did not differ between cows fed CS and AS-K. Increases in dietary RDP usually increase urinary N excretion (Nennich et al, 2006). However, an increase in RDP without altering dietary CP concentration did not affect urinary N excretion in dairy cows (Tacoma et al, 2017;Savari et al, 2018). In the current study, es-timated RDP % of CP was 10.2 and 11.2% for CS and AS-K, respectively (NRC, 2001); therefore, the increase in RDP by 1% unit and numerical decreases in DMI for AS-K versus CS did not affect urinary N excretion.…”

Milk proteome from in silico data aggregation allows the identification of putative biomarkers of negative energy balance in dairy cows

“…The main objective of this computational data aggregation from 20 publications, with 35 datasets (Table 2), is to obtain an overview of milk proteins independently of breed, age, country and methodologies of protein isolation and identification. Methods for protein isolation were density gradient ultracentrifugation 15,39 , centrifugation and washing 10,22,40 , (ultra)centrifugation with acidification 48 and with major proteins depletion 42,45,46 , or (ultra)centrifugation without acidification 13,17,41,43,44,47,49–53 .…”

“…Methods for protein identification were FASP, dimethyl labelling, LC-LTQ-Orbitrap/MS 44 , electrophoresis gel LC-MS/MS 52 , iTRAQ labelling, SCX and LC-MS/MS 10,22,39,43,48 , Maldi-(TOF/TOF)-MS detection 42,47,49,50 and from 1 to 15 repetitions of (nano)LC-MS/MS runs 13,15,17,40,41,45,46,51,53 . Protein identifiers ( ID ) reported in the publications were extracted from tables in Portable Document Format (PDF) or from supplementary data files using Tabula software (www.tabula.technology, Last update February 11, 2017).…”

Bioactive peptides from milk: animal determinants and their implications in human health

“…Milk protein composition is also affected by the ratio between rumen degradable protein (RDP) and rumen undegradable protein (RUP). Tacoma et al (2017) compared the milk composition in cows fed either high RDP:RUP ratio diets (62.4:37.6) or low RDP:RUP ratio diets (48.7:51.3). Cows receiving the high RDP: RUP ratio diet showed increased milk urea N and plasma urea N concentrations (15.7 and 1.02 mmol/l, respectively) than those fed low RDP:RUP diets (14.6 and 0.98 mmol/l, respectively).…”