ABSTRACT
We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal−Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA−Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2−99.8% viability indicated low cell death rates. Proliferation (range, 1.02−1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.
ABSTRACT
In vitro procedures are commonly used to estimate rumen protein degradability and protein digestibility of feed ingredients. However, it is unclear how well these assays correlate to in vivo data. The objectives of this work were to compare postruminal protein availability estimates from one in vitro procedure and one in situ/in vitro procedure with in vivo observations for blood meal (BM), feather meal (FM), and a rumen-protected lysine prototype (RP-Lys). The FM and BM used for this experiment were subsamples of material assessed in vivo by an isotope-based method and the RP-Lys subsamples were of a prototype tested in two in vivo trials: a lactation trial and by plasma appearance. Subsamples of the BM (n = 14) and the FM (n = 22) were sent to each of three different laboratories for in vitro or in situ/in vitro analysis of crude protein (CP) and determination of rumen undegraded protein (RUP) and digested RUP (dRUP). Subsamples of the RP-Lys (n = 5) were sent to one laboratory for in vitro analysis of CP, RUP, and dRUP. Two diets containing BM or FM were assessed using the Cornell Net Carbohydrate and Protein System (CNCPS) v6.55 with ingredient inputs derived from either the CNCPS feed library, the isotope dilution method, or an average of the in vitro results from the three laboratories to determine how much the differences among estimates affected ingredient values. In vitro dRUP estimates for BM from one laboratory closely matched those determined in vivo (66.7% vs. 61.2%, respectively), but no in vitro estimates for FM matched the in vivo values. Surprisingly, there were significant differences in protein digestibility estimates from the modified three-step procedure across the two laboratories for BM (P < 0.0001) and for FM (P < 0.0001) indicating significant variation among laboratories in application of the method. Within all laboratories, BM estimates were reported in a narrow range (CV values of 2.6 or less). However, when testing multiple samples of FM or the RP-Lys prototype, CV values within a laboratory ranged up to 11 and 34, respectively. For the RP-Lys, dRUP estimates from the in vitro method were roughly half of that determined by the in vivo methods suggesting poor concordance between the in vitro and in vivo procedures for this ingredient. The inconsistencies within and among laboratories accompanied with dissimilarities to in vivo data is problematic for application in nutrition models. Additional refinement to the in vitro techniques is warranted.
ABSTRACT
Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.
